This work was supported by the Special Funds for Fundamental Research Funds of institutions of higher-learning affiliated with central departments. We appreciate the support of Classical Prescription Basic Research Team of the Beijing University of Chinese Medicine.

All of the procedures performed in this study were approved by the Ethics Review Committee at the Beijing University of Chinese Medicine (approval number 2017BZYYL00120).
1. Preparation and Characterization of Colloidal Gold
NOTE: For colloidal gold synthesis, as colloidal gold is easily adsorbed on the inner wall of the vessel and is prone to precipitation by impurities, the vessel for synthesis and storage of colloidal gold should be thoroughly cleaned and soaked in acid (40 mL of distilled water, 360 mL of concentrated sulfuric acid, 20 g of potassium dichromate) or subjected to surface passivation treatment. A citric acid reduction method was used to synthesize colloidal gold.
<ol>
	<li>Turn on the magnetic stirrer and place the flask (250 mL) on the mixer.</li>
	<li>Prepare 4% gold chloride acid solution and 1% sodium citrate solution, respectively.</li>
	<li>Add 120 mL of distilled water to a round bottom flask and heat it to boiling on a thermostatic magnetic stirrer.</li>
	<li>Keep boiling, and quickly add 0.5 mL of 4% chloroauric acid and 5 mL of 1% sodium citrate.</li>
	<li>Observe the color of the solution. The pale yellow chloroauric acid solution turns wine red within a few minutes.</li>
	<li>Continue heating for 10 minutes, until the solution changes from colorless to transparent wine red.</li>
	<li>Turn off the power of the thermostatic magnetic mixer, cool to room temperature, and move the mixture to a clean bottle. Store it at 4 &#176;C.</li>
	<li>Determine the size and morphology of the AuNPs by ultraviolet spectroscopy and TEM imaging. 
	&#8203;NOTE: Different sizes of colloid gold particles for various applications can be obtained by changing the proportion of citrate sodium and chloroauric acid.</li>
</ol>
2. Synthesis of AuNPs-mAb Conjugate
NOTE: Since antibodies bind to colloidal gold by electrostatic adsorption, charges on the surface of proteins and colloidal gold directly affect the binding intensity; therefore, the buffer pH value is an important factor affecting the stability of the antibody-colloidal gold conjugate. SSD and anti-SSD mAbs are used as examples in this protocol.
<ol>
	<li>Evaluation of coupling pH
	<ol>
		<li>Add 100 &#956;L of NaCl solution (10% m/v) into eight tubes.</li>
		<li>Adjust the AuNP solutions at pH 5, 6, 7, 8, 9, 10, 11 and 12 with 0.1 M K2CO3.</li>
		<li>Add 100 &#956;L of colloidal gold solution (pH adjusted 5-12) to eight tubes containing NaCl.</li>
		<li>Allow the solutions to stand for several minutes after blending. Observe the color change of each tube solution, and record the tube that remains red.</li>
		<li>Choose the pH value with the least addition of K2CO3 and stable solution color as the optimum pH value for preparing AuNP-mAb conjugates. 
		&#8203;NOTE: Do not use a pH meter because the probe may be destroyed by AuNPs.</li>
	</ol>
	</li>
	<li>Evaluation of antibody amount
	<ol>
		<li>Add 100 &#956;L of NaCl solution (10% m/v) to eight tubes.</li>
		<li>Add 100 &#956;L of colloidal gold solution with optimum pH to each tube.</li>
		<li>Add the monoclonal antibody solutions (protein concentration 0.1 mg/mL-3.2 mg/mL) to the abovementioned eight tubes.</li>
		<li>Allow the solutions to stand for several minutes after blending. Observe the color change of each tube solution, and record the tube that remains red.</li>
		<li>Choose the antibody amount with the lowest concentration of mAb and stable solution color as the optimum mAb amount for preparing AuNP-mAb conjugates.</li>
	</ol>
	</li>
	<li>Prepare the resuspension buffer: add 1 M Tris-HCl (pH 8.8), 1% (w/v) BSA, 0.5% (v/v) Tween 20 and 1% (v/v) PEG 20000 and blend well.</li>
	<li>Synthesis of AuNP-mAb conjugate
	<ol>
		<li>Take 10 mL of colloidal gold solution and use 0.1 M K2CO3 to adjust the solution to the optimal pH value.</li>
		<li>Slowly add SSD mAbs at appropriate concentrations and shake at room temperature for 30 min.</li>
		<li>Centrifuge the mixture at 83 x g (1,000 rpm) for 10 min at 4 &#176;C. Remove the precipitate, which contains impurities or precipitated colloidal gold.</li>
		<li>Centrifuge the mixture at 8,330 x g (10,000 rpm) for 30 min at 4 &#176;C. Discard the supernatant, and the precipitate is the colloidal gold-mAb conjugate.</li>
		<li>Add the resuspension buffer to dissociate the precipitation.</li>
	</ol>
	</li>
</ol>
3. Assembly of the Strip
NOTE: For later flow immunoassays, the selection and pretreatment of membrane material will directly affect the test, which should be investigated. The immunochromatographic strip consists of a sample pad, a conjugate pad, a nitrocellulose (NC) membrane, an absorbent pad and a PVC board (Figure 1). The membrane material should be checked and evaluated by stereomicroscopy to eliminate inhomogeneity.
<ol>
	<li>Paste the NC membrane on a PVC board 2 cm apart from the edge of the suction end of the board.</li>
	<li>Add SSD-BSA (2 mg/mL) dropwise to the NC membrane (2 cm apart from the upper edge) as a test line (1 mm wide), and add goat anti-mouse IgG (1.5 mg/mL) dropwise on the NC membrane (2 cm apart from the lower edge) as a control line (1 mm wide). Control the amount of protein added.</li>
	<li>Attach the absorbent pad to the PVC sheet above the NC membrane and overlap it with the NC membrane by 2 mm.</li>
	<li>Submerge the glass fiber membrane into the AuNPs-mAb conjugate solution. Dry the wet membrane in an incubator at 37 &#176;C.</li>
	<li>Trim the glass fiber membrane to 5 cm long and 2 cm wide and use it as a conjugate pad.</li>
	<li>Paste the pretreated conjugate pad under the NC membrane. The length of overlap with the NC membrane should be 0.1 cm. 
	NOTE: The glass fiber membrane has a strong ability to bind and release proteins.</li>
	<li>Trim the fusion 3 membrane to 1.8 cm long and 3.5 cm wide and use it as the sample pad.</li>
	<li>Paste the sample pad on the PVC board and overlap it with the conjugate pad by 2 mm.</li>
	<li>Cut the assembled paper board into 3.5-mm-wide strips using a cutting machine and compact it using a batch lamination system.</li>
	<li>Finally, place the test strips into the shell, seal them in an aluminum foil bag containing desiccant, and store them away from light. The ICSs are now assembled. 
	&#8203;NOTE: The above is the laboratory procedure. In production, gold spraying equipment and cross-membrane instruments are used to spray gold and make the T and C lines.</li>
</ol>
4. Quantitative Detection
<ol>
	<li>Drop 50 &#181;L of sample solution onto the sample hole to observe the chromatography process. 
	NOTE: As a result of the capillary action driven by the absorbent pad, the sample solution migrates to the other end of the strip. When the sample solution reaches the conjugate pad, the SSD (antigen) in the sample reacts with the AuNPs-mAb preloaded on the pad. When the solution migrates and reaches the T-line, the AuNPs-mAb without SSD can be selectively captured by SSD-BSA (antigen-carrier protein conjugate), showing as a red color on the T-line. Then, the solution migrates to the C line, where the AuNPs-mAb are captured by goat anti-mouse IgG in the region, thus showing as a red color on the C line.</li>
	<li>Analyze the strips with a portable strip reader. The machine can provide the ratio of the test line to the control line (T/C).</li>
	<li>Evaluate the specificity, sensitivity, repeatability and stability of the ICS test. 
	NOTE: Under qualitative detection, one red line indicates a positive result (control line). Two red lines indicate a negative result (test and control lines). If the control line is not present, the test is considered invalid.</li>
</ol>

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The authors have nothing to disclose.

In this work, we present a protocol for the preparation of mAbs against natural product-derived small molecules. The essential steps and the matters needing attention in the procedure have been outlined, and we have demonstrated the utility of this protocol using the small molecule SSD as an example. Example spectra, TEM images, quantitative results and methodological investigations are shown in representative data. Hence, we have demonstrated that the colloidal gold production, AuNP-mAb conjugation and strip assembly st...

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost and rapid detection. The nitrocellulose membrane as the carrier and colloidal gold as markers of immune chromatography rapid diagnostic reagents are the most commonly used POCT (point of care testing) method, and the testing scope of the project is wider. From their original application in monitoring during pregnancy, their use has been extended to monitor blood coagulation state1,2, myocardial injury3, veterinary medicine4, pesticide residues5, infectious diseases6 and drug concentrations. More types of samples can be assessed, including urine, saliva, whole blood, serum and other body fluids7,8,9.
In recent years, numerous novel assays have been developed for detecting biomarkers in the diagnosis of disorders, including HPLC, UPLC, LC-MS and ELISA, which are sensitive and accurate, credible and specific. However, these methods require sophisticated instrumentation, complex preprocessing and time-consuming treatments9. Hence, developing a more rapid and convenient point-of-care diagnostic strategy for the self- and real-time detection of medicinal active compounds is urgent10,11.
The popularity of ICSs, especially for common tests, is driven by their ease of use, as they do not require professionals or elaborate instrumental setups12. In other words, people who do not have special training can operate strips or self-tests13. The results of the test can be obtained in 5 minutes, which means it can be used for site inspections14. Moreover, according to our calculations, the cost of strips could be lower than 1 RMB15, which means that the tests are inexpensive to promote16. Hence, the ICS is a relatively accurate, simple, and inexpensive disposable device. ICSs based on colloidal gold17,18 are also applied in rapid COVID-19 detection.
The principle of ICS can be divided into sandwich ICS and competitive ICS. Figure 1A is a schematic diagram of the sandwich ICS, which is mainly used for detecting macromolecular substances such as proteins, including tumor markers, inflammatory factors, and human chorionic gonadotropin (HCG, early pregnancy antigen). In this method, paired antibodies targeted at different epitopes of the antigen are used, and the capture antibody is dried on the NC membrane as a test line. Labeled antibody is dried on the conjugate pad, and secondary antibody is used as the control line.
Figure 1B is a schematic diagram of competitive ICS, which is mainly used to detect small molecule substances (MWCO &#60; 2000 Da). The coating antigen is fixed on the NC membrane as a test line, and the labeled antibody is dried on the conjugate pad. During detection, the sample and labeled antibody flow through the detection line under capillary action, and the coated antigen competitively binds free antigen in the sample and develops a red color on the detection line.
Recently, we described the procedure of monoclonal antibody generation against natural products19. In this work, we developed a novel lateral flow immunoassay based on the prepared anti-SSD mA20 for rapid, on-site detection. The results indicate that the immunochromatography assay is an indispensable and convenient tool for detecting natural product-derived compounds.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62754/62754fig01.jpg" /> 
Figure 1 Schematic diagram of the immunochromatography assay (A) Sandwich immunochromatographic test strips. (B) Indirect competitive immune chromatographic test strips. This figure has been modified from Zhang et al., 201821. <a href="https://www.jove.com/files/ftp_upload/62754/62754fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Chloroauric acid solution (HAuCl4)</td>
			<td>Tianjin Fu Chen Chemical Reagents Factory</td>
			<td>JY-SJ102</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>AMRESCO</td>
			<td>332</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tube 15 mL</td>
			<td>Corning</td>
			<td>430645</td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge tube 50 mL</td>
			<td>Corning</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>ELISA plates, 96 well</td>
			<td>NUNC</td>
			<td>655101</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Sinopharm</td>
			<td>H5072</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass fibre membranes</td>
			<td>Jieyi</td>
			<td>XQ-Y6</td>
			<td></td>
		</tr>
		<tr>
			<td>goat-anti-mouse IgG antibody</td>
			<td>applygen</td>
			<td>C1308</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membranes</td>
			<td>Millipore</td>
			<td>millipore 180</td>
			<td></td>
		</tr>
		<tr>
			<td>ovalbumin</td>
			<td>Beijing BIODEE</td>
			<td>5008-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>PEG20000</td>
			<td>Sigma Aldrich</td>
			<td>RNBC6325</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 10mL</td>
			<td>COSTAR</td>
			<td>4488</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette 25mL</td>
			<td>FALCON</td>
			<td>357525</td>
			<td></td>
		</tr>
		<tr>
			<td>semi-rigid PVC sheets</td>
			<td>Jieyi</td>
			<td>JY-C104</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium citrate</td>
			<td>Beijing Chemical Works</td>
			<td>C1034</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium periodate</td>
			<td>Sinopharm Chemical</td>
			<td>BW-G0008</td>
			<td></td>
		</tr>
		<tr>
			<td>Sulfo-GMBS</td>
			<td>Perbio Science Germany</td>
			<td>22324</td>
			<td></td>
		</tr>
		<tr>
			<td>TipOne Tips 1,000 &#181;L</td>
			<td>Starlab</td>
			<td>S1111-2021</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>Solarbio</td>
			<td>77-86-1</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Solarbio</td>
			<td>9005-64-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

development of a lateral flow immunochromatographic strip for rapid and quantitative detection of small molecule compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Characterization of colloidal gold 
The prepared colloidal gold solutions were claret red. TEM analyses were used to determine the morphology and shape of AuNPs (Figure 2A-D). Figure 2A and Figure 2B reveal that the particles are polyhedral in shape and uniformly distributed. The average diameter of AuNPs was found to be approximately 14 nm (Figure 2C). A high-reso...

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Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

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Biochemistry

7.1K Views.  Beijing University of Chinese Medicine. Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Video: Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Highly Sensitive and Rapid Fluorescence Detection with a Portable FRET Analyzer

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and amino acids. However, the innate weak signal intensity of FRET sensors is a major challenge that prevents their application in various fields and makes the use of expensive, high-end fluorometers necessary. Previously, we built a cost-effective, high-performance FRET analyzer that can specifically measure the ratio of two emission wavelength bands (530 and 480 nm) to achieve high detection sensitivity. More recently, it was discovered that FRET sensors with bacterial periplasmic binding proteins detect ligands with maximum sensitivity in the critical temperature range of 50 - 55 &#176;C. This report describes a protocol for assessing sugar content in commercially-available beverage samples using our portable FRET analyzer with a temperature-specific FRET sensor. Our results showed that the additional preheating process of the FRET sensor significantly increases the FRET ratio signal, to enable more accurate measurement of sugar content. The custom-made FRET analyzer and sensor were successfully applied to quantify the sugar content in three types of commercial beverages. We anticipate that further size reduction and performance enhancement of the equipment will facilitate the use of hand-held analyzers in environments where high-end equipment is not available.

This protocol describes the rapid and highly sensitive quantification of F&#246;rster resonance energy transfer (FRET) sensor data using a custom-made portable FRET analyzer. The device was used to detect maltose within a critical temperature range that maximizes detection sensitivity, enabling practical and efficient assessment of sugar content.

Recent improvements in F&#246;rster resonance energy transfer (FRET) sensors have enabled their use to detect various small molecules including ions and ...

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies that allow simultaneous measurement of multiple samples have become widespread and greatly reduce experimental costs and times. Our laboratory developed a technique called combined precursor isotopic labeling and isobaric tagging (cPILOT), which enhances sample multiplexing of traditional isotopic labeling or isobaric tagging approaches. Global cPILOT can be applied to samples originating from cells, tissues, bodily fluids, or whole organisms and gives information on relative protein abundances across different sample conditions. cPILOT works by 1) using low pH buffer conditions to selectively dimethylate peptide N-termini and 2) using high pH buffer conditions to label primary amines of lysine residues with commercially-available isobaric reagents (see Table of Materials/Reagents). The degree of sample multiplexing available is dependent on the number of precursor labels used and the isobaric tagging reagent. Here, we present a 12-plex analysis using light and heavy dimethylation combined with six-plex isobaric reagents to analyze 12 samples from mouse tissues in a single analysis. Enhanced multiplexing is helpful for reducing experimental time and cost and more importantly, allowing comparison across many sample conditions (biological replicates, disease stage, drug treatments, genotypes, or longitudinal time-points) with less experimental bias and error. In this work, the global cPILOT approach is used to analyze brain, heart, and liver tissues across biological replicates from an Alzheimer&#39;s disease mouse model and wild-type controls. Global cPILOT can be applied to study other biological processes and adapted to increase sample multiplexing to greater than 20 samples.

Enhanced Sample Multiplexing of Tissues Using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is a quantitative proteomics strategy that enhances sample multiplexing capabilities of isobaric tags. This protocol describes the application of cPILOT to tissues from an Alzheimer's disease mouse model and wild-type controls.

There is an increasing demand to analyze many biological samples for disease understanding and biomarker discovery. Quantitative proteomics strategies ...

Robust Comparison of Protein Levels Across Tissues and Throughout Development Using Standardized Quantitative Western Blotting

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances in both basic and clinical research. However, as with many similar experimental techniques, the outcome of Western blot analyses is easily influenced by choices made in the design and execution of the experiment. Specific housekeeping proteins have traditionally been used to normalize protein levels for quantification, however, these have a number of limitations and have therefore been increasingly criticized over the past few years. Here, we describe a detailed protocol that we have developed to allow us to undertake complex comparisons of protein expression variation across different tissues, mouse models (including disease models), and developmental timepoints. By using a fluorescent total protein stain and introducing the use of an internal loading standard, it is possible to overcome existing limitations in the number of samples that can be compared within experiments and systematically compare protein levels across a range of experimental conditions. This approach expands the use of traditional western blot techniques, thereby allowing researchers to better explore protein expression across different tissues and samples.

This method describes a robust and reproducible approach for the comparison of protein levels in different tissues and at different developmental timepoints using a standardized quantitative western blotting approach.

Western blotting is a technique that is commonly used to detect and quantify protein expression. Over the years, this technique has led to many advances ...

Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall process costs. Alternative, cost-effective capture steps are therefore valuable for industrial-scale manufacturing, where large quantities of a single mAb are produced. Here we present a method for the immobilization of a DsRed-based epitope ligand to a cross-linked agarose resin allowing the selective capture of the HIV-neutralizing antibody 2F5 from crude plant extracts without using Protein A. The linear epitope ELDKWA was first genetically fused to the fluorescent protein DsRed and the fusion protein was expressed in transgenic tobacco (Nicotiana tabacum) plants before purification by immobilized metal-ion affinity chromatography. Furthermore, a method based on activated cross-linked agarose was optimized for high ligand density, efficient coupling and low costs. The pH and buffer composition and the soluble ligand concentration were the most important parameters during the coupling procedure, which was improved using a design-of-experiments approach. The resulting affinity resin was tested for its ability to selectively bind the target mAb in a crude plant extract and the elution buffer was optimized for high mAb recovery, product activity and affinity resin stability. The method can easily be adapted to other antibodies with linear epitopes. The new resins allow gentler elution conditions than Protein A and could also reduce the costs of an initial capture step for mAb production.

In this procedure, a DsRed-based epitope ligand is immobilized to produce a highly selective affinity resin for the capture of monoclonal antibodies from crude plant extracts or cell culture supernatants, as an alternative to Protein A.

The purification of monoclonal antibodies (mAbs) is commonly achieved by Protein A affinity chromatography, which can account for up to 25% of the overall ...

A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...