Z.C.A. was supported by the Scientific and Technological Research Council of Turkey (TUBITAK) 2219- International Postdoctoral Research Fellowship Program. Research reported in this publication was supported by the National Center for Complimentary and Integrative Medicine of the National Institutes of Health under award number 1R41AT011716-01. This work was also partially supported by American Society of Pharmacognosy Research Starter Grant, Regis Technologies grant to L.C. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

1. Cell culture of SH-SY5Y neuroblastoma cells (TrkB and TrkB-NULL (parental) cell lines)
NOTE: Cell lines (SH-SY5Y Cell Line (TrkB, BR6) and SH-SY5Y Parental Cell Line (TrkB NULL))49,50 were purchased from Kerafast. Cultured cells are used as a source of the transmembrane receptors to be immobilized for the preparation of CMAC columns. The following steps describe how to obtain cell membrane fragments and assemble functional CMAC columns.
<ol>
	<li>Grow the cells in a cell culture medium prepared by mixing 450 mL of RPMI media, 50 mL of FBS, 5 mL of penicillin/streptomycin solution, and 0.3 mg/mL of geneticin (G418) in a 150 mm cell culture dish at 37 &#176;C in a humid atmosphere with 5% CO2.</li>
</ol>
2. Cell harvesting
<ol>
	<li>Confirm cell confluency (80%-90%) using a microscope, reached after 3-4 days after cell passaging.</li>
	<li>Aspirate cell culture medium from above the cells and wash the cells twice with phosphate-buffered saline (PBS, 1x, pH 7.4). Remove PBS and add 2 mL of low concentrated trypsin (0.25%) to detach cells.</li>
	<li>Gently swirl the plate to evenly cover all the cells with trypsin solution and incubate the cells for approximately 2 min at 37 &#176;C in a humid atmosphere with 5% CO2. Add 8 mL of cell culture medium to detaching cells and transfer detached cells to a 50 mL conical tube and place it on ice.</li>
	<li>Use a hemocytometer to estimate the number of cells (approximately 3 x 107 cells). Mix cell suspension by pipetting up and down to obtain even cell density in the mixture. Place 10 &#181;L of cell suspension under the coverslip and count the cells under a microscope using a 40x objective.</li>
</ol>
3. Cell homogenization
<ol>
	<li>Prepare stock solutions of the following protease inhibitors: benzamidine (200 mM), phenylmethylsulfonyl fluoride (PMSF, 10 mM), and commercially available protease inhibitors cocktail (100x concentration) containing 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride (AEBSF), aprotinin, bestatin, and leupeptin. 
	CAUTION: PMSF should only be handled inside the fume hood.
	<ol>
		<li>Prepare benzamidine stock solution (200 mM) by dissolving 120 mg of benzamidine in 5 mL of ultrapure deionized water. Store at 4 &#176;C and use within a day. Freshly prepare solution before each use (recommended).</li>
		<li>Prepare PMSF stock solution (10 mM) by dissolving 0.017 g of PMSF in 10 mL of ethanol. Store at - 20 &#176;C.</li>
		<li>Prepare protease inhibitor cocktail (100x) by dissolving commercially available protease inhibitor mixture in 1 mL of ultrapure deionized water. Mix thoroughly and aliquot 200-300 &#181;L of the cocktail and store at - 20 &#176;C before use.</li>
	</ol>
	</li>
	<li>Prepare ATP stock solution (100 mM) by dissolving 55.114 mg of ATP disodium salt hydrate in 1 mL of deionized ultrapure water. Mix thoroughly and aliquot 100 &#181;L of the mixture and store at - 20 &#176;C before use.</li>
	<li>Prepare buffer by dissolving 3.03 g of tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, 50 mM), 2.9 g of sodium chloride (NaCl, 100 mM), 0.22 g of calcium chloride anhydrous (CaCl2, 3 mM), 0.2 g of magnesium chloride hexahydrate (MgCl2, 2 mM) and 0.19 of potassium chloride (KCl, 5 mM) in 500 mL of ultrapure deionized water.</li>
	<li>Prepare homogenization buffer by mixing 17.3 mL of the buffer prepared in step 3.3 with 0.3 mL (3 mM) of benzamidine stock solution, 0.2 mL (0.1 mM) of PMSF stock solution, 0.2 mL of protease inhibitor cocktail mixture, 20 &#181;L (100 &#181;M) of ATP stock solution, 2 mL (10%) of glycerol, and 0.029 g (5mM) of EDTA (Table 1). Adjust pH to 7.4 using hydrochloric acid solution.</li>
	<li>Spin down the cells harvested in step 2.3 at 4 &#176;C for 5 min at 400 x g. Remove the supernatant and wash the remaining cell pellet with 10 mL of ice-cold PBS (1x, pH 7.4). Spin down the cells again at 4 &#176;C for 5 min at 400 x g.</li>
	<li>Discard the supernatant and replace it with 20 mL of homogenization buffer prepared in step 3.4. Place the conical tube on ice.</li>
	<li>Transfer the cell suspension into a 40 mL Dounce homogenizer tissue grinder and place it on ice. Homogenize the suspension manually, on ice, using 40 up-and-down pestle strokes. Transfer homogenized cell suspension into a 50 mL conical tube.</li>
	<li>Centrifuge the homogenate at 4 &#176;C for 7 min at 400 x g. Discard the pellet and transfer the supernatant to a new conical tube and centrifuge it at 4 &#176;C for 30 min at 47900 x g. Save the cell membrane pellet and discard the supernatant.</li>
</ol>
4. Cell membrane solubilization
<ol>
	<li>Prepare solubilization buffer by mixing 8.7 mL of the buffer solution made in step 3.3 with 0.1 mL (0.1 mM) of PMSF stock solution, 0.15 mL (3 mM) of benzamidine stock solution, 0.1 mL of protease inhibitor cocktail, 10 &#181;L (100 &#181;M) of ATP stock solution, 1 mL (10%) of glycerol and 0.2 g (2%) of sodium cholate (Table 2).</li>
	<li>Transfer the solubilization buffer into the conical tube with cell membrane fragments obtained in step 3.8 and resuspend the pellet. Rotate the resulting mixture at 150 rpm at 4 &#176;C for 18 h. 
	&#8203;NOTE: At this point, the experiment can be paused for overnight cell membrane pellet solubilization.</li>
</ol>
5. Cell membrane immobilization on IAM.PC.DD2 particles
<ol>
	<li>After 18 h, centrifuge the solubilization mixture at 4 &#176;C for 30 min at 47900 x g.</li>
	<li>Keep the supernatant and discard the pellet. Add 100 mg of IAM.PC.DD2 particles, to the supernatant, vortex and rotate the resulting suspension mixture at 150 rpm at 4 &#176;C for 1 h.</li>
	<li>To facilitate the immobilization of cell membrane fragments on the IAM.PC.DD2 particles proceed to the dialysis step as outlined below.
	<ol>
		<li>Prepare dialysis buffer in 4 L of ultrapure deionized water by adding 24 g of tris-HCl (50 mM), 23.4 g of NaCl (100 mM), 0.06 g of CaCl2 (0.1 mM), 5.85 g of EDTA (5 mM), and 0.07 g of PMSF (0.1 mM; Table 3). 
		&#8203;NOTE: PMSF is not soluble in water, therefore dissolve it first in small volume of ethanol and then add slowly into the beaker with the buffer.</li>
		<li>Adjust pH to 7.4 with hydrochloric acid solution. Place the buffer at 4 &#176;C for a minimum of 1 h before proceeding to the dialysis step.</li>
		<li>Prepare dialysis tube by cutting 10 cm of cellulose membrane dialysis tubing (10 K MWCO, 35 mm) and transfer the suspension containing IAM.PC.DD2 particles and cell membrane fragments into the dialysis tube. Use dialysis tubing clips to close both ends of the dialysis tube.</li>
		<li>Place the dialysis tube containing the IAM.PC.DD2 stationary phase in the dialysis buffer and dialyze at 4 &#176;C for 24 h while gently stirring.</li>
		<li>After 24 h, place the dialysis tubing in the freshly prepared dialysis buffer and continue dialysis for another 24 h.</li>
	</ol>
	</li>
</ol>
6. CMAC column packing
<ol>
	<li>Prepare ammonium acetate buffer (10 mM, pH 7.4) that will be used to wash the IAM.PC.DD2 particles and in column characterization experiments by dissolving 0.7708 g of ammonium acetate in 1 L of ultrapure deionized water. Adjust pH to 7.4 with hydrochloric acid solution.</li>
	<li>After 48 h of dialysis, remove the dialysis tube from the dialysis buffer and transfer the content of the dialysis tube into a 15 mL conical tube.</li>
	<li>Centrifuge the mixture at 4 &#176;C for 5 min at 400 x g. Discard the supernatant and wash the remaining pellet three times with 10 mL of ammonium acetate buffer, centrifuging the mixture at 4 &#176;C for 5 min at 400 x g, after each wash.</li>
	<li>After the third wash, resuspend the remaining pellet in 1 mL of ammonium acetate buffer. Mix it thoroughly and use the resulting slurry to pack the 5/20 glass column to yield CMAC chromatography column.</li>
	<li>To pack the column, first place a bottom filter previously soaked with ammonium acetate buffer, into the filter holder. Fit the filter holder in the glass column and screw the column cap to secure the position of the holder. Place the column vertically in a finger clamp and secure it in the lab stand. Place a beaker below the column.</li>
	<li>Using a single channel pipettor transfer a small volume of the slurry obtained in step 6.4 into the glass column. Pour the material slowly, holding the pipette tip against the glass column wall. Allow the packing material to settle before pouring another volume of slurry.</li>
	<li>To speed up the packing process remove the buffer from above the stationary bed using a micropipette between each step. Repeat these steps until 1 mL of the slurry is packed.</li>
	<li>Place a top filter and screw the adapter unit so that there is no remaining buffer above the stationary phase, as presented in Figure 1. Secure the position of the adapter unit with the adapter lock.</li>
	<li>Connect the column to a high-performance liquid chromatography (HPLC) pump, set the flow rate to 0.2 mL/min, and wash the column overnight with ammonium acetate buffer. The column is now ready for the characterization step. Store the column at 4 &#176;C until use. 
	NOTE: For longer storage (column not used for longer than a week) run the column with 0.05% sodium azide solution in ammonium acetate buffer and store at 4 &#176;C. 
	&#8203;CAUTION: Sodium azide is highly toxic when ingested orally or absorbed through the skin; it should only be handled under the fume hood. Make sure to wear a lab coat, safety glasses, and gloves (nitrile preferred) when working with sodium azide.</li>
</ol>
7. CMAC column characterization
<ol>
	<li>Confocal microscopy and BDNF binding
	<ol>
		<li>Prepare IAM.PCC.DD2 stationary phase particles with immobilized cell membrane fragments obtained separately from SH-SY5Y neuroblastoma cells overexpressing TrkB and SH-SY5Y TrkB-NULL cells by following the instructions from step 1 to step 6.4.</li>
		<li>For samples that will be incubated with BDNF prior to antibody staining, prepare BDNF stock solution by dissolving 10 &#956;g of BDNF in 100 &#956;L of ultrapure deionized water. Store the solution at -20 &#176;C.
		<ol>
			<li>Transfer in separate 1.5 mL microcentrifuge tubes, 100 &#956;L aliquot of IAM.PC.DD2 column packing material with immobilized SH-SY5Y Neuroblastoma cells overexpressing TrkB and 100 &#956;L aliquot of IAM.PC.DD2 column packing material with immobilized SH-SY5Y TrkB-NULL cells suspended in ammonium acetate buffer.</li>
			<li>Add 390 &#956;L of ammonium acetate buffer, 10 &#956;L of BDNF stock solution, and 0.5 &#956;L of ATP stock solution (prepared in step 3.2) into each tube and incubate for 1 h at room temperature with rocking.</li>
		</ol>
		</li>
		<li>For samples that will not be incubated with BDNF prior to antibody staining, transfer 100 &#956;L aliquot of IAM.PC.DD2 column packing material with immobilized SH-SY5Y Neuroblastoma cells overexpressing TrkB suspended in ammonium acetate into 1.5 mL microcentrifuge tubes.</li>
		<li>Add 400 &#956;L of ammonium acetate buffer and 0.5 &#956;L of ATP stock solution (prepared in step 3.2) into each tube and incubate for 1 h at room temperature with rocking.</li>
		<li>Spin the samples prepared in steps 7.1.2 and 7.1.4 at 4 &#176;C for 1 min at 10,000 x g and discard the supernatant.</li>
		<li>Wash the resulting pellets with 500 &#956;L of ammonium acetate buffer for 10 min and spin again at 4 &#176;C for 1 min at 10,000 x g and discard the supernatant.</li>
		<li>To each of the pellets, add 220 &#956;L of ammonium acetate buffer, 25 &#956;L of 10% normal goat serum, and 5 &#956;L of primary anti-BDNF antibody. Incubate in a cold room (4 &#176;C) overnight with rocking.</li>
		<li>Upon completion of the incubation step spin the mixture for 1 min at 10,000 x g at 4 &#176;C and discard the supernatant.</li>
		<li>Wash the resulting pellet 3 times with ammonium acetate buffer containing 1% mild detergent (e.g., sodium cholate) for 10 min and spin the mixtures for 1 min at 10,000 x g at 4 &#176;C and discard the supernatant.</li>
		<li>Prepare secondary antibody solution by mixing 900 &#956;L of ammonium acetate buffer, 100 &#956;L of 10% normal goat serum, and 1 &#956;L of fluorophore-conjugated secondary antibody (1:1,000 dilution). Add 300 &#956;L of secondary antibody solution to each of the pellets obtained in step 7.1.9. and incubate overnight at 4 &#176;C with rocking.</li>
		<li>Spin the mixture at 10,000 x g for 1 min at 4 &#176;C and discard the supernatant.</li>
		<li>Wash the resulting pellet 3 times with ammonium acetate buffer containing 1% mild detergent (e.g., sodium cholate) for 10 min and spin the mixture for 1 min at 10,000 x g at 4 &#176;C and discard the supernatant.</li>
		<li>Resuspend the resulting pellets in 50 &#956;L of ammonium acetate buffer. Place 20 &#956;L of each of the mixtures on a slide and cover with a coverslip.</li>
		<li>Image the IAM.PC.DD2 particles with the immobilized cell membrane fragments using a confocal laser scanning microscope with excitation at 488 nm and emission at 520 nm generated using a solid state laser system. Use a 20x objective with a NA of 0.75 and WD of 0.35mm.</li>
	</ol>
	</li>
	<li>Frontal affinity chromatography using 7,8-DHF as a marker ligand
	<ol>
		<li>Connect a CMAC column to an HPLC system with a diode-array detector and/or mass spectrometer.</li>
		<li>Prepare 500 mL of 1 mM solution of 7,8-DHF in ammonium acetate buffer containing 5% methanol.</li>
		<li>Pump constant concentration of the marker ligand (1 mM) through the CMAC column at 0.4 mL/min flow rate at room temperature. Monitor the elution profile using either a diode array detection (DAD) (wavelength 254 nm) detector or mass spectrometer (use single ion monitoring mode; m/z 253 in negative ionization mode).</li>
		<li>After completion of the run perform overnight wash by running ammonium acetate buffer through the column.</li>
		<li>Repeat steps 7.2.2. - 7.2.4. using different concentrations of the marker ligand (750 nM, 500 nM, 300 nM), washing the columns overnight after each run. Use frontal affinity chromatography to calculate ligand Kd, as reviewed in detail, elsewhere.24,51</li>
	</ol>
	</li>
	<li>Displacement studies with Centella asiatica&#160;(L.) Urb.(gotu kola) extract
	<ol>
		<li>Prepare 500 mL of 500 nM solution of 7,8-DHF in ammonium acetate buffer containing 5% methanol and 0.2% aqueous gotu kola extract (10 mg/mL).</li>
		<li>Pump constant concentration of the marker ligand (500 nM) with the extract through the column at 0.4 mL/min flow rate at room temperature. Monitor the elution profile using either a DAD detector (wavelength 254 nm) or a mass spectrometer (use single ion monitoring mode; m/z 253 in negative ionization mode).</li>
		<li>After completion of the run perform overnight wash by running ammonium acetate buffer through the column.</li>
		<li>Plot the elution profiles for 500 nM 7,8-DHF and the one obtained after running the solution with gotu kola extract to inspect for marker ligand displacement.</li>
	</ol>
	</li>
</ol>
8. Missing peak chromatography approach to identify potential TrkB binders from gotu kola extract
<ol>
	<li>Fractionation of gotu kola extract on CMAC columns
	<ol>
		<li>Connect separately the CMAC TrkB and TrkB-NULL columns to an HPLC system and inject 50 &#956;L of the aqueous gotu kola extract (10 mg/mL) on each of the columns. Pump ammonium acetate buffer (10 mM, pH 7.4) containing 5% methanol through the column at 0.4 mL/min flow rate at room temperature.</li>
		<li>Collect fractions separately from CMAC TrkB and TrkB-NULL columns as follows: 0-5 min, 5-10 min, 10-15 min, 15-20 min, 20-25 min, 25-30 min, 30-35 min, 35-40 min, 40-45 min, 45-50 min, 50-55 min, 55-60 min.</li>
		<li>Freeze and lyophilize the obtained fractions. Resuspend lyophilized fractions in 50 &#956;L of methanol before proceeding to ultra-performance liquid chromatography and mass spectrometry analysis.</li>
	</ol>
	</li>
	<li>Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) analysis of gotu kola CMAC fractions
	<ol>
		<li>Analyze all the fractions obtained in point 8.1.3. using a mass spectrometer coupled with a UPLC system (UPLC-MSE analysis mode). Analyze the fractions using a C18 column (2.1 x 50 mm, 1.7 &#181;m) with a C18 VanGuard pre-column (2.1 mm x 5 mm, 1.7 &#181;m).</li>
		<li>Elute the column using the following gradient solutions at a flow rate of 0.3 mL/min with mobile phase A (water with 0.1% formic acid) and B (acetonitrile with 0.1% formic acid): 0.0 min 99% A 1% B, 1.5 min 84% A 16% B, 5.0 min 80% A 20% B, 7.0 min 75% A 25% B, 10.0 min 65% A 35% B, 20.0-24.0 min 1% A 99% B, 25.0-29.0 min 99% A 1% B.</li>
		<li>Set the injection volume for each sample to 3 &#181;L. Perform MSE in resolution positive and negative ion modes, with collision energy at 4V for low energy and 20-35 V for high energy.</li>
		<li>Use the following ESI source conditions in positive ionization mode: capillary voltage 1.5 kV, sampling cone 40 V, source offset 80, source temperature 100 &#176;C, desolvation temp. 350 &#176;C, cone gas flow 38.0 L/h, desolvation gas 400 L/h.</li>
		<li>Use the following ESI source conditions in negative ionization mode: capillary voltage 1.45 kV, sampling cone 40 V, source offset 80, source temperature 110 &#176;C, desolvation temp. 300 &#176;C, cone gas flow 50.0 L/h, desolvation gas flow 652 L/h.</li>
	</ol>
	</li>
</ol>

Lukasz Ciesla collaborates with Regis Technologies, the provider of the IAM.PC.DD2 particles.

Identification of active compounds present in complex mixtures of specialized metabolites is a very challenging task23. Traditionally, individual compounds are isolated, and their activity is tested in different assays. This approach is time-consuming and costly and often leads to isolation and identification of the most abundant and well-characterized compounds23.&#160;Currently used high-throughput screening assays rely heavily on screening combinatorial chemistry librari...

Botanical mixtures are rich in pharmacologically active compounds1, serving as a good source for the identification of new drug hits and leads2,3,4,5.&#160;The discovery of new medicines from natural products has been a fruitful approach and many currently approved drugs originated from compounds first identified in nature. The chemical diversity of natural compounds is hard to be matched by man-made libraries of chemically synthesized molecules. Many natural compounds interact with and modulate human protein targets and can be considered evolutionarily optimized drug-like molecules6. These natural compounds are particularly well suited for drug lead identification to use in neurological disorders6. Two of the currently FDA-approved drugs for the management of Alzheimer&#39;s disease (AD) are derived from natural alkaloids, namely: galantamine and rivastigmine (a derivative of physostigmine)6. L-DOPA, presently the most commonly prescribed drug for Parkinson&#39;s disease, was first identified from the broad bean (Vicia faba L.)7. Pergolide and lisuride, dopaminergic receptor agonists are the derivatives of natural ergot alkaloids from the parasitic fungus Claviceps purpurea8. Reserpine, an alkaloid isolated from Indian snakeroot (Rauvolfia serpentina (L.) Benth. ex Kurz)&#160;was one of the first antipsychotic drugs9. Recently, dysregulated immune response and systemic inflammation have been linked to the development of numerous neurological ailments, such as major depressive disorder or neurodegenerative diseases10. A plant-based diet together with other lifestyle interventions has been found to improve cognitive and functional abilities in the elderly11,12,13,14,15,16,17,18,19,20,21. Certain electrophilic molecules belonging to triterpenes and polyphenols have been found to modulate inflammatory responses in both in vitro and in vivo models12. For instance, natural compounds containing &#945;,&#946;-unsaturated carbonyl (e.g., curcumin, cinnamaldehyde), or isothiocyanate group (e.g., sulforaphane) interfere with Toll-like receptor-4 (TLR4) dimerization inhibiting the downstream synthesis of pro-inflammatory cytokines in a murine interleukin-3 dependent pro-B cell line12,22. Epidemiological evidence points strongly that dietary phytochemicals, present in complex food matrices, may also constitute a viable source of new drug leads6.
One of the major obstacles in the identification of biologically active molecules present in plant extracts, including plant-based food, is the complexity of the investigated samples. Traditionally, the individual compounds are isolated, purified, and subsequently tested for biological activity. This approach usually leads to the identification of the most abundant and well-characterized compounds. Phenotypic drug discovery approaches without a defined molecular target rely on the bio-guided-fractionation of complex mixtures23. In this approach, an extract is fractionated into less complex sub-fractions that are subsequently tested in phenotypic assays. The isolation and purification of active compounds are guided by biological activity verified in the assay. The knowledge of the identity of a specified drug target may significantly speed up the identification of pharmacologically active compounds present in complex mixtures. Those approaches are usually based on the immobilization of the molecular target, for example, an enzyme, on a solid surface, like magnetic beads23. The immobilized targets are subsequently used in the screening experiments resulting in the isolation of compounds interacting with the target. While this approach has been extensively used in the identification of compounds targeting cytosolic proteins, it has been less commonly applied in the identification of chemicals interacting with transmembrane proteins (TMPs)23. An additional challenge in the immobilization of TMPs stems from the fact that the activity of the protein depends on its interaction with cell membrane phospholipids and other molecules in the bilayer such as cholesterol23,24. It is important to preserve these subtle interactions between proteins and their native phospholipid bilayer environment when attempting to immobilize the transmembrane target.
In cellular membrane affinity chromatography (CMAC) cell membrane fragments, and not purified proteins, are immobilized on the artificial membrane (IAM) stationary phase particles23. IAM stationary phases are prepared by covalently bonding phosphatidylcholine analogs onto silica. Recently novel classes of IAM stationary phases have been developed in which free amine and silanol groups are end-capped (IAM.PC.DD2 particles). During CMAC columns preparation cell membrane fragments are immobilized onto the surface of IAM particles through adsorption.
CMAC columns have been used to date to immobilize different classes of TMPs including ion channels (e.g., nicotinic receptors), GPCRs (e.g., opioid receptors), protein transporters (e.g., p-glycoprotein), etc.24. The immobilized protein targets have been used in the characterization of pharmacodynamics (e.g., dissociation constant, Kd) or determining binding kinetics (kon and koff) of small molecule ligands interacting with the target as well as in the process of identification of potential new drug leads present in complex matrices24. Here we present the preparation of CMAC columns with the immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders.
Previous studies showed that the activation of the brain-derived neurotrophic factor (BDNF)/TrkB pathway is associated with the improvement of certain neurological ailments, such as AD or major depressive disorder25,26,27,28. It was reported that BDNF levels and its receptor TrkB expression decrease in AD, and similar reductions impair hippocampal function in animal models of AD29. Decreased levels of BDNF were reported in serum and brain of AD patients30,31,32. Tau overexpression or hyperphosphorylation were found to down-regulate BDNF expression in primary neurons and AD animal models33,34,35. Additionally, BDNF was reported to have protective effects on &#946;-amyloid induced neurotoxicity in vitro and in vivo36. Direct BDNF administration into the rat brain was shown to increase learning and memory in cognitively impaired animals37. BDNF/TrkB emerged as a valid target for ameliorating neurological and psychiatric disorders including AD28,38. Targeting the BDNF/TrkB signaling pathway for the development of therapies in AD will potentially enhance our understanding of the disease39. Unfortunately, BDNF itself cannot be used as a treatment because of its poor pharmacokinetic properties and adverse side effects40. Small molecule activators of TrkB/BDNF pathways have been explored as potential TrkB ligands41,42,43. Among tested small molecule agonists, 7,8-dihydroxyflavone (7,8-DHF), has been shown to activate the BDNF/TrkB pathway41,44,45,46. A derivative of 7,8-DHF (R13; 4-Oxo-2-phenyl-4H-chromene-7,8-diyl bis(methylcarbamate)) is currently under consideration as a possible drug for AD47. Recently, it was shown that several antidepressants work through directly binding to TrkB and promoting BDNF signaling, further stressing the importance of pursuing TrkB as a valid target to treat various neurological disorders48.
The protocol describes the process of assembling functional TrkB column and TrkB-NULL negative control column. The columns are characterized using a known natural product small-molecular ligand: 7,8-DHF. Additionally, we describe the process of screening complex matrices, using plant extract as an example, for the identification of compounds interacting with TrkB.

<table><tbody><tr><td>7-8 Dihydroxyflavone hydrate</td><td>Sigma-Aldrich</td><td>D5446-10 mg</td><td>&amp;ge;98% (HPLC)</td></tr><tr><td>Adenosine 5&#039;-triphosphate (ATP) disodium salt hydrate</td><td>Sigma-Aldrich</td><td>A2383-1 g</td><td></td></tr><tr><td>Ammonium acetate</td><td>VWR Chemicals BDH</td><td>BDH9204-500 g</td><td></td></tr><tr><td>BDNF antibody</td><td>Invitrogen</td><td>PA5-15198-400 &amp;mu;L</td><td>Primary antibody; 2 mg/mL of concentration</td></tr><tr><td>Benzamidine hydrochloride hydrate</td><td>Sigma-Aldrich</td><td>B6506-25 g</td><td></td></tr><tr><td>Brain derived neurotrophic factor (BDNF) human</td><td>Sigma-Aldrich</td><td>B3795-10 &amp;mu;g</td><td>Recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture</td></tr><tr><td>Calcium chloride</td><td>VWR Analytical</td><td>BDH9224-1 kg</td><td></td></tr><tr><td>Cholic acid sodium salt</td><td>Alfa Aesar</td><td>J62050-100 g</td><td></td></tr><tr><td>Dounce homogenizer</td><td>VWR</td><td>71000-516</td><td>40 mL, 285 mm (overall lenght), 32 x 140 mm (O.D. x H)</td></tr><tr><td>Ethanol</td><td>Sigma-Aldrich</td><td>493511</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>VWR Analytical</td><td>BDH-9232-500 g</td><td></td></tr><tr><td>Fetal bovine serum</td><td>Sigma-Aldrich</td><td>F2442-500 mL</td><td>sterile-filtered, suitable for cell culture</td></tr><tr><td>G418 disulfate salt solution</td><td>Sigma-Aldrich</td><td>G8168-100 mL</td><td>50 mg/mL in H2O, 0.1 &amp;mu;m filtered, suitable for cell culture</td></tr><tr><td>Glycerol</td><td>VWR Life Science</td><td>E520-100 mL</td><td></td></tr><tr><td>Immobilized artificial membrane (IAM.PC.DD2)</td><td>Regis Technologies, Inc.</td><td>1-771050-500</td><td></td></tr><tr><td>Magnesium chloride hexahydrate</td><td>VWR Analytical</td><td>BDH9244-500 mL</td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>322425</td><td></td></tr><tr><td>Nikon C2 DUVb</td><td>Nikon</td><td></td><td>Confocal laser scanning microscope</td></tr><tr><td>Normal goat serum (10%)</td><td>Life Technologies</td><td>50197Z</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma-Aldrich</td><td>P4333-100 mL</td><td></td></tr><tr><td>Phenylmethanesulfonyl fluoride (PMSF)</td><td>Thermo Scientific</td><td>36978-5 g</td><td></td></tr><tr><td>Phosphate buffered saline (PBS)</td><td>VWR Life Science</td><td>K812-500 mL</td><td>1x</td></tr><tr><td>Potassium chloride</td><td>VWR Chemicals BDH</td><td>0395-1 kg</td><td></td></tr><tr><td>Protease inhibitor cocktail</td><td>VWR Life Science Ambreso</td><td>M221-1 mL</td><td>Proteomics grade, containing 50 mM AEBSF, 30 &amp;micro;M aprotonin, 1 mM bestatin, 1 mM E-64 and 1 mM leupeptin</td></tr><tr><td>RPMI-1640 medium</td><td>Sigma-Aldrich</td><td>R8758-500 mL</td><td>with L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture</td></tr><tr><td>Secondary antibody goat anti-rabbit IgG (H+L)</td><td>Invitrogen Alexa Flour Plus 488</td><td>A32731</td><td></td></tr><tr><td>SH-SY5Y Neuroblastoma cell lines expressing Trk-B</td><td>Kerafast</td><td>ECP007</td><td></td></tr><tr><td>SH-SY5Y Trk-NULL cell line</td><td>Kerafast</td><td>ECP005</td><td></td></tr><tr><td>Snake skin dialysis tubing</td><td>Thermo Scientific</td><td>88245</td><td>10K MWCO, 35 mm dry I.D.</td></tr><tr><td>Sodium azide</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium chloride</td><td>BDH VWR Analytical</td><td>BDH9286-2.5 kg</td><td></td></tr><tr><td>Tricorn 5/20 column</td><td>GE Healthcare</td><td>24-4064-08</td><td></td></tr><tr><td>Tris-HCl</td><td>VWR Life Science</td><td>0497-1 kg</td><td></td></tr><tr><td>Trypsin-EDTA solution</td><td>Sigma-Aldrich</td><td>T4049-500 mL</td><td>0.25%, sterile-filtered, suitable for cell culture, 2.5 g porcine trypsin and 0.2&amp;nbsp;g EDTA</td></tr></tbody></table>

cellular membrane affinity chromatography columns to identify specialized plant metabolites interacting with immobilized tropomyosin kinase receptor b

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, penicillin, paclitaxel, rapamycin, or artemisinin, commonly used in medical practice, have been first identified and isolated from natural products. However, the identification and isolation of biologically active specialized metabolites from natural sources is a challenging and time-consuming process. Traditionally, individual metabolites are isolated and purified from complex mixtures, following the extraction of biomass. Subsequently, the isolated molecules are tested in functional assays to verify their biological activity. Here we present the use of cellular membrane affinity chromatography (CMAC) columns to identify biologically active compounds directly from complex mixtures. CMAC columns allow for the identification of compounds interacting with immobilized functional transmembrane proteins (TMPs) embedded in their native phospholipid bilayer environment. This is a targeted approach, which requires knowing the TMP whose activity one intends to modulate with the newly identified small molecule drug candidate. In this protocol, we present an approach to prepare CMAC columns with immobilized tropomyosin kinase receptor B (TrkB), which has emerged as a viable target for drug discovery for numerous nervous system disorders. In this article, we provide a detailed protocol to assemble the CMAC column with immobilized TrkB receptors using neuroblastoma cell lines overexpressing TrkB receptors. We further present the approach to investigate the functionality of the column and its use in the identification of specialized plant metabolites interacting with TrkB receptors.

Following the protocol, two CMAC chromatographic columns were assembled: one with the immobilized SH-SY5Y neuroblastoma cell membrane fragments with overexpressed TrkB and one with SH-SY5Y TrkB-NULL cell membrane fragments. The correctly assembled CMAC column is presented in Figure 1 and the steps involved in cell membrane fragment immobilization are presented in Figure 2.
Since the immobilization of TrkB receptors on IAM.PC.DD2 chrom...

Watch this Scientific Journal Video about Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B at JoVE

Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

The protocol describes the preparation of cell membrane affinity chromatography (CMAC) columns with immobilized cell membrane fragments containing functional transmembrane tropomyosin kinase receptor B proteins. The use of CMAC columns in the identification of specialized plant metabolites interacting with these receptors and present in complex natural mixtures is also explained.

Chemicals synthesized by plants, fungi, bacteria, and marine invertebrates have been a rich source of new drug hits and leads. Medicines such as statins, ...

cellular-membrane-affinity-chromatography-columns-to-identify

Research

JoVE Journal

Biochemistry

2.5K Views.  The University of Alabama. Deze verticale presenteert belangrijke stappen in het samenstellen van celmembraanaffiniteitschromatografiekolommen met functionele transmembraanreceptoren. Deze kolommen kunnen worden gebruikt voor de identificatie van kleine moleculen die aanwezig zijn in complexe extracten en interageren met geïmmobiliseerde transmembraanreceptoren. Cellulaire membraanaffiniteitschromatografiekolommen versnellen de identificatie van biologisch actieve verbindingen die aanwezig zijn in complexe mengsels.

Video: Cellular Membrane Affinity Chromatography Columns to Identify Specialized Plant Metabolites Interacting with Immobilized Tropomyosin Kinase Receptor B

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

Chemistry

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Affinity Purification of Chloroplast Translocon Protein Complexes Using the TAP Tag

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the translocon at the outer (TOC) and inner (TIC) chloroplast membranes. The TOC and TIC complexes are multimeric and probably contain yet unknown components. One of the main goals in the field is to establish the complete inventory of TOC and TIC components. For the isolation of TOC-TIC complexes and the identification of new components, the preprotein receptor TOC159 has been modified N-terminally by the addition of the tandem affinity purification (TAP) tag resulting in TAP-TOC159. The TAP-tag is designed for two sequential affinity purification steps (hence "tandem affinity"). The TAP-tag used in these studies consists of a N-terminal IgG-binding domain derived from Staphylococcus aureus Protein A (ProtA) followed by a calmodulin-binding peptide (CBP). Between these two affinity tags, a tobacco etch virus (TEV) protease cleavage site has been included. Therefore, TEV protease can be used for gentle elution of TOC159-containing complexes after binding to IgG beads. In the protocol presented here, the second Calmodulin-affinity purification step was omitted. The purification protocol starts with the preparation and solubilization of total cellular membranes. After the detergent-treatment, the solubilized membrane proteins are incubated with IgG beads for the immunoisolation of TAP-TOC159-containing complexes. Upon binding and extensive washing, TAP-TOC159 containing complexes are cleaved and released from the IgG beads using the TEV protease whereby the S. aureus IgG-binding domain is removed. Western blotting of the isolated TOC159-containing complexes can be used to confirm the presence of known or suspected TOC and TIC proteins. More importantly, the TOC159-containing complexes have been used successfully to identify new components of the TOC and TIC complexes by mass spectrometry. The protocol that we present potentially allows the efficient isolation of any membrane-bound protein complex to be used for the identification of yet unknown components by mass spectrometry.

We here present a proven and tested protocol for the purification of chloroplast protein import complexes (TOC-TIC complex) using the TAP-tag. The one-step affinity-isolation protocol can potentially be applied to any protein and be used to identify new interaction partners by mass spectrometry.

Chloroplast biogenesis requires the import of thousands of nucleus-encoded proteins into the plastid. The import of these proteins depends on the ...

Classification of Tropane Alkaloids from Plant Metabolites Using LC-MS/MS

Applications of Liquid-Chromatography Tandem Mass Spectrometry in Natural Products Research: Tropane Alkaloids as a Case Study

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and can yield promising leads for resistant or emerging diseases. The challenge, however, is twofold: not only must researchers find natural products and elucidate their structures, but they must also identify what is worth isolating and assaying (and what is already known - a process known as dereplication). With the advent of modern analytical instrumentation, the pace of natural product discovery and dereplication has accelerated. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has become an especially valuable technique for identifying and classifying chemical structures. Tropane alkaloids (TAs) are plant-derived compounds of great medicinal and toxicological significance. In this study, we developed an LC-MS/MS-based screening workflow utilizing the multiple MS/MS configurations available on a triple-quadrupole (QQQ) mass spectrometer to annotate and classify TA structures based on their distinct fragmentation patterns. By using a combination of data-dependent (DD) product ion scans, precursor ion scans (PrIS), and neutral loss scans (NLS), we applied this method to TA-rich extracts of the nightshades Datura stramonium and Datura metel. This method is rapid, sensitive, and was successfully employed for both preliminary dereplication of complex TA-containing samples and for the discovery of a novel candidate for isolation, purification (and eventual bioassay).

Author Spotlight: Discovering New Alkaloids in Plants with Advanced Mass Spectrometry Techniques

We present a method for rapid mass spectrometry (MS)/mass spectrometry (MS)-based annotation and classification of tropane alkaloids, useful for both preliminary dereplication of tropane-containing samples and discovery of novel alkaloids for isolation.

Although many drugs utilized today are synthetic in origin, natural products still provide a rich source of novel chemical diversity and bioactivity, and ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

Biology

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Spatiotemporal Investigation of ERL1 Dynamics via Confocal Imaging

Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane system. This includes the secretory transport of newly synthesized proteins to the cell surface or the outside of the cell, the endocytic transport of extracellular cargoes or plasma membrane components into the cell, and the recycling or shuttling transport of cargoes between the subcellular organelles, etc. Membrane trafficking events are crucial to the development, growth, and environmental adaptation of all eukaryotic cells and, thus, are under stringent regulation. Cell-surface receptor kinases, which perceive ligand signals from the extracellular space, undergo both secretory and endocytic transport. Commonly used approaches to study the membrane trafficking events using a plasma membrane-localized leucine-rich-repeat receptor kinase, ERL1, are described here. The approaches include plant material preparation, pharmacological treatment, and confocal imaging setup. To monitor the spatiotemporal regulation of ERL1, this study describes the co-localization analysis between ERL1 and a multi-vesicular body marker protein, RFP-Ara7, the time series analysis of these two proteins, and the z-stack analysis of ERL1-YFP treated with the membrane trafficking inhibitors brefeldin A and wortmannin.

Author Spotlight: Image-Based Methods to Study Membrane Trafficking Events in Stomatal Lineage Cells  

Several commonly used methods are introduced here to study the membrane trafficking events of a plasma membrane receptor kinase. This manuscript describes detailed protocols including the plant material preparation, pharmacological treatment, and confocal imaging setup.

In eukaryotic cells, membrane components, including proteins and lipids, are spatiotemporally transported to their destination within the endomembrane ...

Chromatography-based Biomolecule Purification Methods

In biochemistry, chromatography-based purification methods are employed to isolate compounds from a complex mixture. Two such methods used commonly by biochemists are size-exclusion chromatography and affinity chromatography. In size-exclusion chromatography, a column packed with porous beads separates components of a mixture based on size. On the other hand, affinity chromatography allows for a more specific separation of biomolecules by using a column that is composed of stationary phase, which contains target-specific ligands.
This video serves as an introduction to size-exclusion and affinity chromatography, as well as the concepts that govern them. A step-by-step procedure for the purification of a histidine-tagged protein by immobilized metal affinity chromatography is described. Applications for both of these chromatographic methods in biochemistry and biomedical research are also profiled.
<div class="chapter_text" id="script_0">
&#34;Chromatography&#34; refers to a wide range of methods used to isolate a component from a complex mixture, an essential step before a biomolecule&#39;s properties and activities can be determined. Each chromatographic technique has a different mechanism for separation, depending on the sample matrix and target compound. This video will focus on the principles and operation of two methods common to biochemistry: size-exclusion and affinity chromatography.
</div>
<div class="chapter_text" id="script_1">
Size-exclusion chromatography or SEC is based on the size of the compounds in the sample. A mobile phase containing the sample is added to a column with a porous material: the stationary phase. The molecules in the sample fall into 1 of 3 categories.
Molecules too large to enter the pores travel the shortest distance through the column. Any species with a molecular weight above this &#34;exclusion limit&#34; will exit the column at the same time. Molecules small enough to freely enter the pores will be retained the longest, and will exit the column together. The molecular weight allowing complete pore entry is the &#34;permeation limit&#34;.
Only molecules between these limits will be separated from one another, as they spend varying amounts of time diffusing into and out of the pores. Smaller molecules are retained longer on the column because they spend more time in the stationary phase, whereas larger molecules within these limits exit earlier.
Molecular weights across 1 to 2 orders of magnitude fall within these limits. Columns are chosen with this in mind, or multiple columns can be used in series if there is a wide range of desired compounds.
Now that you&#39;ve seen the theory of SEC, let&#39;s look at how it is carried out.
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To begin the SEC procedure, the column must be equilibrated with deionized water and chromatography buffer. Once prepared, buffer containing the sample is injected onto the column. The buffer is then pushed through at a low flow rate. A detector monitors what exits the column to determine the presence of the desired analyte. Large molecules with molecular weights above the exclusion limit exit the column at the same time. Small fractions from the column are collected in tubes. Each fraction is tested for the quality of the target molecule by gel electrophoresis or other analytical techniques.
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Now let&#39;s have a look at affinity chromatography or AC, one of the most efficient ways to purify proteins. Many biomolecules bind selectively to certain ligands-a property which AC utilizes by adhering a target-specific ligand to the stationary phase.
When the mixture flows through the column, the target molecules attach to the ligand, and the rest flow through. After the mixture has passed through the column, the target molecule can be collected through one of two elution methods based on specificity.
Biospecific elution can be said to a have a &#34;normal-&#34; or &#34;reverse-role&#34;. In normal-role biospecific elution, an agent is added that competes with the adhered ligand to bind with the target biomolecule.
In reverse-role biospecific elution, an agent competes with the target to bind to the adhered ligand. The second elution type, nonspecific elution, lowers the target-to-ligand binding by changing the solution&#39;s pH, ionic strength, or polarity. If a protein does not bind to a ligand that can be immobilized, the protein can be expressed containing a &#34;tag&#34;: short peptide sequences engineered to bind to the ligand.
One variety is immobilized metal ion affinity chromatography, &#34;IMAC&#34; for short, where an adhered metal ligand, like nickel or cobalt, binds to histidine residues on the modified protein. Through molecular biology techniques, target proteins are generated with repeating histidine residues, called a polyhistidine-tag, which binds to the metal via the imidazole side chain on histidine. Once bound, the protein can be collected with free imidazole via reverse-role specific elution and later used in a wide variety of downstream applications.&#160;Now that you&#39;ve seen the theory of affinity chromatography, let&#39;s look at an IMAC procedure in the laboratory.

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In IMAC, the stationary phase can be added directly to the mixture of mobile phase and sample, allowing the binding of the his-tagged protein. This slurry is then poured into the column, where the non-bound compounds drip into waste, while the slurry remains. The slurry container is rinsed to collect residual resin and sample, which is added to the column.
The resin is stirred to ensure the unbound components are free-flowing. Added wash buffer helps flush them away. Once all of the unbound components have been removed, the waste is replaced with a container to collect the target protein.
Buffer containing imidazole is added, stirred, and allowed to rest to unbind the target molecule. The imidazole binds to the metal, replacing and releasing the tagged protein. The freed protein is collected, and the imidazole step is repeated to ensure total collection. To further purify the sample, SEC can be run on the sample prior to analysis.
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Now that we&#39;ve seen the theory and procedure of these two techniques, let&#39;s look at some of the ways they&#39;re applied in the biochemical field.
A common reason to purify proteins is to study their role in disease. Cystic fibrosis is caused by defects in the cystic fibrosis transmembrane conductance regulator protein, or CFTR. After growing the protein with a his-tag in yeast, both affinity and size-exclusion chromatography allow the isolation of the protein, followed by the study of its function.
In some instances, the presence of a polyhistidine tag can change a protein&#39;s structure, thereby affecting its function. Another common tag is maltose-binding protein or MBP, which will bind to bound amylose in a column. Maltose is then used to release the complex. The MBP can then be cleaved and removed with SEC to produce the pure desired protein.
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You&#39;ve just watched JoVE&#39;s video on size-exclusion and affinity chromatography. It covered the theory of the techniques, went over general procedures, and covered some of the uses of the techniques.
Thanks for watching!
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