Name: study of the functions and activities of neuronal k-cl co-transporter kcc2 using western blotting
Uploaded: 2022-12-09T13:15:00
Description: Watch this Scientific Journal Video about Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting at JoVE.com

This work was supported by The Royal Society UK (Grant no. IEC\NSFC\201094), and a Commonwealth Ph.D. Scholarship.

NOTE: The protocol describes the western blotting method to detect specific proteins of interest.
1. Cell culture and transfection
<ol>
	<li>Warm all the reagents in the bead bath (37 &#730;C) before the cell culture procedure. Prepare culture medium, Dulbecco&#39;s Modified Eagle Medium (DMEM), supplemented with 10% fetal bovine serum, 1% each of 2mM L-glutamine, 100x non-essential amino acid, 100 mM sodium pyruvate, and 100 units/mL penicillin-streptomycin.</li>
	<li>Thaw ...

Many methods have been used to measure the activities of SLC12 of CCCs that are expressed in the neurons, including KCC2. Many of these techniques have proven to enhance scientific knowledge on the analysis of the functional relevance of these transporters and their structure-function patterns in different disease-related mutations. Critically, there are advantages and caveats to the various methods21. However, the protocol explained above, outlined how to assess KCC2 phosphorylation at kinase reg...

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition1,2,3,4. The maintenance of low intraneuronal Cl- concentration ([Cl-]i) at 4-6 mM by KCC2 facilitates &#947;-aminobutyric acid (GABA)/glycine hyperpolarization and synaptic inhibition in the brain and spinal cord5. Failure in the proper regulation of KCC2 has been associated with the prevalence of several neurological diseases, including epilepsy4. Furthermore, decreased KCC2-mediated Cl&#8722; extrusion and impaired hyperpolarizing GABAA and/or glycine receptor-mediated currents have been implicated in epilepsy, neuropathic pain, and spasticity6,7. Neuronal KCC2 is negatively modulated via phosphorylation of key regulatory residues within its C-terminal intracellular domain by the with-no-lysine (WNK)-STE20/SPS1-related proline/alanine-rich (SPAK)/Oxidative stress-responsive (OSR) kinase signaling complex1, which facilitates the maintenance of depolarized GABA activity in immature neurons2,8,9. The WNK-SPAK/OSR1 phosphorylates threonine residues 906 and 1007 (Thr906/Thr1007) and subsequently downregulates mRNA gene expression of KCC2, leading to a consequent deterioration in its physiological function8,10. More importantly, however, it is already a fact that the WNK-SPAK/OSR1 kinase complex is known to phosphorylate and inhibit KCC2 expression1,2,4,11,12, and that the inhibition of the kinase complex signaling pathways to phosphorylate Thr906/Thr1007 has been linked with the increased expression of the KCC2 mRNA gene13,14,15. It is important to note that the regulation of neuronal KCC2 and Na+-K+-2Cl- cotransporters 1 (NKCC1) expression via protein phosphorylation works concomitantly and in converse patterns1,4,16.
There has been consistent and considerable progress with regard to the understanding of mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enables researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. The protocol presented here highlights the application of western blotting techniques to study the functions and activities of neuronal K+-Cl- co-transporter KCC2 by investigating the phosphorylation of the cotransporter at kinase regulatory sites Thr906/1007.
Western blot is a method used to detect specific proteins of interest from a sample of tissue or cell. This method first separates the proteins by size through electrophoresis. The proteins are then electrophoretically transferred to a solid support (usually a membrane) before the target protein is marked using a specific antibody. The antibodies are conjugated to different tags or fluorophore-conjugated antibodies that are detected using either colorimetric, chemiluminescence, or fluorescence methods. This allows for a specific target protein to be detected from a mixture of proteins. This technique has been used to characterize phosphospecific sites of KCCs1 and has been used to identify kinase inhibitors that inhibit KCC3 Thr991/Thr1048 phosphorylation17.By following this protocol, one can specifically detect total and phosphorylated KCC2 from cell/tissue lysates. In principle, the detection of protein-conjugated antibodies by this technique is highly instrumental as it helps to improve the understanding of cooperative activities at the phospho-sites of KCC2, which sheds light on the molecular mechanisms involved in their physiological regulations. The quantitative analysis of the total protein expression is representative of the function and activity of KCC2. There are other classical methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>40% acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A2917</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Ammonium Per Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>248614</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>anti pSPAK</td>
			<td>Dundee University</td>
			<td>S670B</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2</td>
			<td>Dundee University</td>
			<td>S700C</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2 pSer940</td>
			<td>Thermo Fisher Scientific</td>
			<td>PA5-95678</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2 pThr1007</td>
			<td>Dundee University</td>
			<td>S961C</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-KCC2 pThr906</td>
			<td>Dundee University</td>
			<td>S959C</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-mouse</td>
			<td>Cell Signalling technology</td>
			<td>66002</td>
			<td>Used as secondary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-NKCC1</td>
			<td>Dundee University</td>
			<td>S841B</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-NKCC1 pThr203/207/212</td>
			<td>Dundee University</td>
			<td>S763B</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-rabbit</td>
			<td>Cell Signalling technology</td>
			<td>C29F4</td>
			<td>Used as secondary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-sheep</td>
			<td>abcam</td>
			<td>ab6900</td>
			<td>Used as secondary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-SPAK</td>
			<td>Dundee University</td>
			<td>S669D</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>anti-&#946;-Tubulin III</td>
			<td>Sigma-Aldrich</td>
			<td>T8578</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>Benzamine</td>
			<td>Merck UK</td>
			<td>135828</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Beta-mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td>Used as component of loading buffer and lysis buffer</td>
		</tr>
		<tr>
			<td>Bradford Coomasie</td>
			<td>Thermo Scientific</td>
			<td>1856209</td>
			<td>Used for lysate protein quantification</td>
		</tr>
		<tr>
			<td>Casting apparatus</td>
			<td>Atto&#160;</td>
			<td>WSE-1165W</td>
			<td>Used to run SDS-page electrophoresis</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804</td>
			<td>Used in lysate preparation</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>VWR</td>
			<td>MicroStar 17R</td>
			<td>Used for spinning samples</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2650-100ML</td>
			<td>Used for cell culture experiment</td>
		</tr>
		<tr>
			<td>Dried Skimmed Milk</td>
			<td>Marvel</td>
			<td>N/A</td>
			<td>Used to make blocking buffer</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium - high glucose</td>
			<td>Sigma-Aldrich</td>
			<td>D6429</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>ECL reagent</td>
			<td>Perkin Elmer</td>
			<td>ORTT755/2655</td>
			<td>Used to develop image for western blotting</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher Scientific</td>
			<td>D/0700/53</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>e4378</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Electrophoresis Power Supply</td>
			<td>BioRad</td>
			<td>PowerPAC HC</td>
			<td>To supply power to run SDS-page electrophoresis</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>ThermoFisher</td>
			<td>E/0650DF/17</td>
			<td>Used for preparing sterilized equipments and environment</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum -&#160; heat inactivated</td>
			<td>Merck Life Sciences UK</td>
			<td>F9665</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Fumehood</td>
			<td>Walker</td>
			<td>A7277</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Gel Blotting - Whatman</td>
			<td>GE Healthcare&#160;</td>
			<td>10426981</td>
			<td>Used in western blotting to make transfer sandwich</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Sigma-Aldrich</td>
			<td>15527</td>
			<td>Used to make buffers</td>
		</tr>
		<tr>
			<td>GraphPad Prism Software</td>
			<td>GraphPad Software, Inc., USA</td>
			<td>Version 6.0</td>
			<td>Used for plotting graphs and analysing data for&#160; western blotting</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Acros Organics</td>
			<td>10647282</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Heating block</td>
			<td>Grant</td>
			<td>QBT1</td>
			<td>Used to heat WB loading samples</td>
		</tr>
		<tr>
			<td>HEK293 cells</td>
			<td>Merck UK</td>
			<td>12022001-1VL</td>
			<td>Cell line for culture experiment</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>Wayne Rasband and Contributors; NIH, USA&#160;</td>
			<td>ImageJ 1.53e</td>
			<td>Used to measure band intensities from western blotting images</td>
		</tr>
		<tr>
			<td>Imaging system</td>
			<td>BioRad</td>
			<td>ChemiDoc MP</td>
			<td>Used to take western blotting images</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>LEEC</td>
			<td>LEEC precision 190D</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Honeywell</td>
			<td>24137</td>
			<td>Used in casting gel for electrophoresis</td>
		</tr>
		<tr>
			<td>L-glutamine solution</td>
			<td>Sigma-Aldrich</td>
			<td>G7513</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Lithium dodecyl sulfate (LDS)</td>
			<td>Novex</td>
			<td>NP0008</td>
			<td>Used as loading buffer for western blotting</td>
		</tr>
		<tr>
			<td>MEM Non-essential amino acid&#160;</td>
			<td>Merck Life Sciences UK</td>
			<td>M7145</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5418</td>
			<td>Used for preparing lysates for WB</td>
		</tr>
		<tr>
			<td>Microplate reader</td>
			<td>BioRad</td>
			<td>iMark</td>
			<td>Used for lysate protein concentration readout</td>
		</tr>
		<tr>
			<td>Microsoft Powerpoint</td>
			<td>Microsoft, USA</td>
			<td>PowerPoint2016</td>
			<td>Used to edit western blotting images</td>
		</tr>
		<tr>
			<td>Molecular Weight Marker</td>
			<td>BioRad</td>
			<td>1610373</td>
			<td>Used for western blotting</td>
		</tr>
		<tr>
			<td>N-ethylmaleimide</td>
			<td>Thermo Fisher Scientific</td>
			<td>23030</td>
			<td>Used for cell culture experiment</td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Fisher Scientific</td>
			<td>45004091</td>
			<td>Used for western blotting</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Gibco</td>
			<td>15140122</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>pH Meter</td>
			<td>Mettler Toledo</td>
			<td>Seven compact s210</td>
			<td>Used to monitor pH of buffer solutions</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonylfluoride (PMSF)</td>
			<td>Sigma-Aldrich</td>
			<td>P7626</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>PKC&#948; pThr505</td>
			<td>Cell Signalling technology</td>
			<td>9374</td>
			<td>Used as primary antibody for western blotting</td>
		</tr>
		<tr>
			<td>Sepharose Protein G</td>
			<td>Generon</td>
			<td>PG50-00-0002</td>
			<td>Used for immunoprecipitation</td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Used as component of wash buffer</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>S7653</td>
			<td>Used to prepare TBS-T buffer</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>L5750</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>sodium orthovanadate</td>
			<td>Sigma-Aldrich</td>
			<td>S6508</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>Sigma-Aldrich</td>
			<td>S8636</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>sodium-&#946;-glycerophosphate</td>
			<td>Merck UK</td>
			<td>G9422</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Staurosporine (from Streptomyces sp.)</td>
			<td>Scientific Laboratory Supplies, UK</td>
			<td>S4400-1MG</td>
			<td>Used for cell culture experiment</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Scientifc Laboratory Supplies</td>
			<td>S0389</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Sigma-Aldrich</td>
			<td>T7024</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Transfer Chamber</td>
			<td>BioRad</td>
			<td>1658005EDU</td>
			<td>Used in western blotting to transfer protein on membrane</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Sigma-Aldrich</td>
			<td>T6066</td>
			<td>Used to make seperating and stacking gel for SDS-PAGE&#160;</td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Used as component of lysis buffer</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA Solution</td>
			<td>Merck Life Sciences UK</td>
			<td>T4049</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P3179</td>
			<td>Used as make TBS-T buffer</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Charles Austen</td>
			<td>Dymax 5</td>
			<td>Used for cell culture</td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td>Scientific Industries</td>
			<td>K-550-GE</td>
			<td>Used in sample preparation</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Scientific Industries Ltd</td>
			<td>Vortex-Genie&#160; K-550-GE</td>
			<td>Used of mixing resolved sample</td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>Grant Instruments Ltd. (JB Academy)</td>
			<td>JBA5</td>
			<td>Used to incubate solutions</td>
		</tr>
	</tbody>
</table>

study of the functions and activities of neuronal k-cl co-transporter kcc2 using western blotting

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively in the neuron and is essential for the proper functioning of Cl- homeostasis and consequently functional GABAergic inhibition. Failure in proper regulation of KCC2 is deleterious and has been associated with the prevalence of several neurological diseases, including epilepsy. There has been considerable progress with regard to understanding the mechanisms involved in the regulation of KCC2, accredited to the development of techniques that enable researchers to study its functions and activities; either via direct (assessing kinase regulatory sites phosphorylation) or indirect (observing and monitoring GABA activity) investigations. Here, the protocol highlights how to investigate KCC2 phosphorylation at kinase regulatory sites - Thr906 and Thr1007- using western blotting technique. There are other classic methods used to directly measure KCC2 activity, such as rubidium ion and thallium ion uptake assay. Further techniques such as patch-clamp-electrophysiology are used to measure GABA activity; hence, indirectly reflecting activated and/or inactivated KCC2 as informed by the assessment of intracellular chloride ion homeostasis. A few of these additional techniques will be briefly discussed in this manuscript.

Here, the representative result presented in Figure 1 investigated the impact of staurosporine and NEM on WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1 in HEK293 cell lines stably expressing KCC2b (HEKrnKCC2b)18 using the western blotting technique. Comprehensive details on the representative results are discussed in Zhang et al.15. Similar to NEM, staurosporine is a broad kinase inhibitor that can enhance KCC2 tr...

Watch this Scientific Journal Video about Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting at JoVE.com

Study of the Functions and Activities of Neuronal K-Cl Co-Transporter KCC2 Using Western Blotting

The present protocol highlights the application of western blotting technique to study the functions and activities of neuronal K-Cl co-transporter KCC2. The protocol describes the investigation of KCC2 phosphorylation at kinase regulatory sites Thr906/1007 via western blotting. Also, additional methods to confirm KCC2 activity are briefly highlighted in this text.

Potassium chloride cotransporters 2 (KCC2) is a member of the solute carrier family 12 (SLC12) of cation-chloride-cotransporters (CCCs), found exclusively ...

This protocol can help to understand the mechanisms involved in KCC2 regulation by directly assessing its kinase molecular size phosphorylation, thus shedding light on its physiological functions and activities. It can be used to identify and characterize molecules like proteins of interest from complex mixtures of related molecules even when a protein expression is very minute. The technique is an essential tool for protein analysis of complex systems and identifying potential mechanisms underlying aberrant tissue function or disease.Demonstrating the procedures will be Sunday Solomon Josiah, a PhD student from my laboratory. Begin by pre-arming all the reagents in the 37 degrees Celsius bead bath and thawing the stably transected rat KCC2b human embryonic kidney 293 cells completely in the bead bath. Then transfer the cells from the cryo vial tube into a centrifuge tube containing five milliliters of fresh media and centrifuge the cells at 1, 200 G for three to five minutes.After aspirating the supernatant using an aspirator pipette fixed to a vacuum pump, resuspend the cells in 10 milliliters of fresh media. Transfer the suspension to a 10 centimeter dish plate and allow it to grow for 48 hours in an incubator having 37 degrees Celsius temperature and 5%carbon dioxide. Once the cells attain 90%confluency, split them by aspirating the old media and rinsing them with two milliliters of PBS.Add two milliliters of trypsin and incubate them for about one to two minutes at room temperature. Wash the trypsinized cells using two milliliters of the complete media. Add nine milliliters of fresh media to the new dishes.Then add one milliliter of solution from the old dish to the new dish. Transfer the split cell dishes to the incubator for 48 hours to attain more than 90%confluency. Treat the cells with either dimethyl sulfoxide, eight micromolar staurosporine, or 0.5 millimolar N-ethylmaleimide for 15 minutes before harvesting in lysis buffer.Aspirate the media from the transected culture dish, place the culture dishes on ice, and wash the cells with ice cold PBS. Aspirate the PBS and add 1.0 milliliters of ice cold lysis buffer to the dish. After scraping the cells from the bottom of the dish using a cold plastic cell scraper, transfer the cell suspension into a microcentrifuge tube placed on ice, followed by constant agitation for 30 minutes at four degrees Celsius.After centrifuging the cell lysates in a cold centrifuge, place it on ice. Collect the supernatant into a pre-cooled fresh tube, and discard the pellet. Pipette 300 microliters of Protein G Sepharose into a microcentrifuge tube.Then centrifuge the solution at 500 G for two minutes. After discarding the supernatant, add 500 microliters of PBS and vortex well. Discard the supernatant after centrifugation and repeat the step.Next, mix one milligram of anti-KCC2 threonine 906 and anti-KCC2 threonine 1007 antibodies with 200 microliters of Protein G Sepharose beads before making up the volume to 500 microliters with PBS. After shaking on a vibrating platform or rotating wheel for two hours at four degrees Celsius, repeat two washes with PBS. Carry out protein quantification on the cell lysates and add one milligram of the cell lysate to the washed beads and incubate in an end-to-end rotator before spinning down.Give three washes with PBS containing 150 millimolar sodium chloride, followed by three washes with 200 microliters of PBS before resuspending the final pellet in 100 microliters of LDS sample buffer. Shake the tubes in a rotor shaker at room temperature for five minutes. Incubate them in a heating block and centrifuge them before using the supernatant for gel loading.Assemble the Western Blot casting apparatus and pour freshly prepared 8%separating gel to cast the gel, allowing about two centimeters of space from the top of the casting glass. Add 200 microliters of absolute isopropanol to the setup and allow it to stand at room temperature for 60 minutes. Remove the isopropanol using a pipette and carefully rinse the gel with about 200 microliters of distilled water.After adding freshly prepared 6%stacking gel to the casting setup, gently fit in the well comb and allow to stand at room temperature for 30 minutes. Then fix the casted gel into the electrophoresis tank. After pouring the running buffer into the tank, load five microliters of molecular weight marker into the first well and an equal amount of protein into each well of the SDS-PAGE gel.Fill up the empty wells with LDS and run the gel for about 90 to 120 minutes at 120 volts. Rehydrate the nitrocellulose membrane with transfer buffer containing 20%methanol. Rinse the gel and membrane with the transfer buffer and gently spread them out on the preparing stack.Arrange the sandwich to be transferred in this order:negative electrode, sandwich foam, filter paper rinsed SDS-PAGE gel, rinsed nitrocellulose membrane, filter paper, sandwich foam, positive electrode. Once done, stack the assembled sandwich in the transfer tank and run at 90 volts for 90 minutes or 30 volts for 360 minutes. Block the dry membrane for one hour at room temperature using a blocking buffer.Incubate the membranes with appropriate dilutions of primary antibody and beta-actin in blocking buffer for one hour at room temperature or overnight at four degrees Celsius. Then give three washes of TBST for five minutes each. Incubate the washed membrane with a secondary antibody diluted in a blocking buffer for 60 minutes and repeat three washes of TBST.Once done, place the washed membrane on the imaging board. To develop the signals, spread the solution prepared by mixing equal volumes of each enhanced chemiluminescence reagent on the membrane before transferring the imaging board to the imaging system for imaging. The treatment of HEK293 cells with staurosporine and N-ethylmaleimide or NEM caused decreased phosphorylation at SPAK target sites, threonine 233 situated in the T-loop kinase domain, and the S-loop phosphorylation site serine 373 of endogenously expressed SPAK.Staurosporine reduced the phosphorylation of serine 940 in HEK294 cells stably expressing KCC2b, but NEM significantly increased its phosphorylation. Furthermore, staurosporine decreases phosphorylation at the threonine 505 site, whereas NEM caused a slight but insignificant increase in phosphorylation at the same site. The differential effects of both compounds on the phosphorylation of protein kinase C at the threonine 505 site and KCC2b at the serine 940 site correlate well.The representative result also showed that NEM caused a significant increase in the expression of the total KCC2 amount, whereas the expression of the total NKCC1 and SPAK were not significantly changed when treated with both compounds. Furthermore, the two compounds caused a decreased phosphorylation of SPAK at threonine 233 and serine 373 sites, and this correlated with the abridged phosphorylation of threonine 906/1007 and threonine 203/207/212 and endogenously expressed NKCC1 respectively. Additionally, staurosporine and NEM reduced and increased the phosphorylation of protein kinase C at the serine 940 site and stably expressed KCC2b respectively, which correlated with the reduction and increment in the phosphorylation of protein kinase C delta at the threonine 505 site upon staurosporine and NEM treatment respectively.If an improper primary or secondary antibody is used, the band will not be seen during the imaging. Also, the signal may not be visible if a too low concentration of antibody is used. The technique is a relevant tool in quantitative analysis of protein and has allowed its use for many scientific and resource purposes, including as an effective early diagnostic tool.

study-functions-activities-neuronal-k-cl-co-transporter-kcc2-using

Research

JoVE Journal

Biochemistry

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating these studies, reincorporation of detergent-solubilized membrane proteins into liposomes is a stochastic process where protein topology is impossible to enforce. This paper offers an alternative method to these challenging techniques that utilizes a liposome-based scaffold. Protein solubility is enhanced by deletion of the transmembrane domain, and these amino acids are replaced with a tethering moiety, such as a His-tag. This tether interacts with an anchoring group (Ni2+ coordinated by nitrilotriacetic acid (NTA(Ni2+)) for His-tagged proteins), which enforces a uniform protein topology at the surface of the liposome. An example is presented wherein the interaction between Dynamin-related protein 1 (Drp1) with an integral membrane protein, Mitochondrial Fission Factor (Mff), was investigated using this scaffold liposome method. In this work, we have demonstrated the ability of Mff to efficiently recruit soluble Drp1 to the surface of liposomes, which stimulated its GTPase activity. Moreover, Drp1 was able to tubulate the Mff-decorated lipid template in the presence of specific lipids. This example demonstrates the effectiveness of scaffold liposomes using structural and functional assays and highlights the role of Mff in regulating Drp1 activity.

Using Scaffold Liposomes to Reconstitute Lipid-proximal Protein-protein Interactions In Vitro

This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.

Studies of integral membrane proteins in vitro are frequently complicated by the presence of a hydrophobic transmembrane domain. Further complicating ...

SorLA and CLC:CLF-1-dependent Downregulation of CNTFR&#945; as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored CNTFR&#945; to form a trimeric complex that subsequently recruits glycoprotein 130/Leukemia Inhibitory Factor Receptor-&#946; (gp130/LIFR&#946;) for signaling. Both CLC and CNTFR&#945; are necessary for signaling but so far CLF-1 has only been known as a putative facilitator of CLC secretion. However, it has recently been shown that CLF-1 contains three binding sites: one for CLC; one for CNTFR&#945; (that may promote assembly of the trimeric complex); and one for the endocytic receptor sorLA. The latter site provides high affinity binding of CLF-1, CLC:CLF-1, as well as the trimeric (CLC:CLF-1:CNTFR&#945;) complex to sorLA, and in sorLA-expressing cells the soluble ligands CLF-1 and CLC:CLF-1 are rapidly taken up and internalized. In cells co-expressing CNTFR&#945; and sorLA, CNTFR&#945; first binds CLC:CLF-1 to form a membrane-associated trimeric complex, but it also connects to sorLA via the free sorLA-binding site in CLF-1. As a result, CNTFR&#945;, which has no capacity for endocytosis on its own, is tugged along and internalized by the sorLA-mediated endocytosis of CLC:CLF-1.
The present protocol describes the experimental procedures used to demonstrate i) the sorLA-mediated and CLC:CLF-1-dependent downregulation of surface-membrane CNTFR&#945; expression; ii) sorLA-mediated endocytosis and lysosomal targeting of CNTFR&#945;; and iii) the lowered cellular response to CLC:CLF-1-stimulation upon sorLA-mediated downregulation of CNTFR&#945;.

SorLA and CLC:CLF-1-dependent Downregulation of CNTFR&amp;#945; as Demonstrated by Western Blotting, Inhibition of Lysosomal Enzymes, and Immunocytochemistry

The present protocol describes how Western blotting and immunocytochemistry combined with inhibition of lysosomal enzymes can be used to demonstrate the downregulation of Ciliary Neurotrophic Factor Receptor-&#945; (CNTFR&#945;), which is conveyed by the interaction between Cytokine-like Factor-1 (CLF-1) and the endocytic receptor sorLA.

The heterodimeric cytokine Cardiotrophin-like Cytokine:Cytokine-like Factor-1 (CLC:CLF-1) targets the glycosylphosphatidylinositol (GPI)-anchored ...

A Western Blotting Protocol for Small Numbers of Hematopoietic Stem Cells

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western blotting protocol was optimized and suitable for the analysis of small numbers of HSCs (500 - 15,000 cells). Phenotypic HSCs were purified, accurately counted, and directly lysed in Laemmli sample buffer. Lysates containing equal numbers of cells were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the blot was prepared and processed following standard Western blotting protocols. Using this protocol, 2,000 - 5,000 HSCs can be routinely analyzed, and in some cases data can be obtained from as few as 500 cells, compared to the 20,000 to 40,000 cells reported in most publications. This protocol should be generally applicable to other hematopoietic cells, and enables the routine analysis of small numbers of cells using standard laboratory procedures.

A standard Western blotting protocol was optimized for analyzing as few as 500 hematopoietic stem or progenitor cells. Optimization involves careful handling of the cell sample, limiting transfers between tubes, and directly lysing the cells in Laemmli sample buffer.

Hematopoietic stem cells (HSCs) are rare cells, with the mouse bone marrow containing only ~25,000 phenotypic long term repopulating HSCs. A Western ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

A11-positive &#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer is widely accepted as the leading neurotoxin in the progress of Alzheimer&#39;s disease (AD) and is considered to be the crucial event in the pathogenesis of AD. Therefore, A&#946; oligomer inhibitors might prevent neurodegeneration and have the potential to be developed as disease-modifying treatments of AD. However, different formation protocols of A&#946; oligomer might lead to oligomers with different characteristics. Moreover, there are not many methods to effectively screen A&#946;1-42 oligomer inhibitors. An A11 antibody can react with a subset of toxic A&#946;1-42 oligomer with anti-parallel &#946;-sheet structures. In this protocol, we describe how to prepare an A11-positive A&#946;1-42 oligomer-rich sample from a synthetic A&#946;1-42 peptide in vitro and to evaluate relative amounts of A11-positive A&#946;1-42 oligomer in samples by a dot blotting analysis using A11 and A&#946;1-42-specific 6E10 antibodies. Using this protocol, inhibitors of A11-positive A&#946;1-42 oligomer can also be screened from semi-quantitative experimental results.

A11-positive &amp;#946;-amyloid Oligomer Preparation and Assessment Using Dot Blotting Analysis

This protocol describes how to prepare A&#946; oligomers from a synthetic peptide in vitro and to evaluate relative amounts of A&#946; oligomer by a dot blotting analysis.

&#946;-amyloid (A&#946;) is a hydrophobic peptide with an intrinsic tendency to self-assemble into aggregates. Among various aggregates, A&#946; oligomer ...

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine whether there is an imbalance in age-related energy metabolism between mitochondrial oxidative phosphorylation and aerobic glycolysis. Here, we show the utilization of commercial colorimetric assay kits for lactate and pyruvate in the model organism C. elegans. Recently, the sensitivity and accuracy of the colorimetric/fluorimetric assay kits have been improved greatly by the research and development conducted by reagent manufacturers. The improved reagents have enabled the use of small-scale assays with a 96-well plate in C. elegans. In general, a fluorimetric assay is superior in sensitivity to a colorimetric assay; however, the colorimetric approach is more suitable for the use in common laboratories. Another important issue in these assays for quantitative determination is protein precipitation of homogenized C. elegans samples. In our protein precipitation method, common precipitants (e.g., trichloroacetic acid, perchloric acid and metaphosphoric acid) are used for sample preparation. A protein-free assay sample is prepared by directly adding cold precipitant (final concentration of 5%) during homogenization.

Small-Scale Colorimetric Assays of Intracellular Lactate and Pyruvate in the Nematode Caenorhabditis elegans

We describe a modified small-scale extraction and colorimetric assays of lactate and pyruvate in the nematode C. elegans. When utilizing commercial assay kits, the technical development of their sensitivity and accuracy is important. Protein precipitation in extraction is the most critical step for the quantitative determination of intracellular metabolites.

Lactate and pyruvate are key intermediates of intracellular energy metabolic pathways. Monitoring the lactate/pyruvate ratio in cells helps to determine ...

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

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 ...

Ultra-High-Speed Western Blot using Immunoreaction Enhancing Technology

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and abundance of specific proteins as well as post-translational protein modifications. This technique has a rich history and remains in widespread use due to its simplicity. However, the western blotting procedure famously takes hours, even days, to complete, with a critical bottleneck being the long incubation times that limit its throughput. These incubation steps are required due to the slow diffusion of antibodies from the bulk solution to the immobilized antigens on the membrane: the antibody concentration near the membrane is much lower than the bulk concentration. Here, we present an innovation that dramatically reduces these incubation intervals by improving antigen binding via cyclic draining and replenishing (CDR) of the antibody solution. We also utilized an immunoreaction enhancing technology to preserve the sensitivity of the assay. A combination of the CDR method with a commercial immunoreaction enhancing agent boosted the output signal and substantially reduced the antibody incubation time. The resulting ultra-high-speed western blot can be accomplished in 20 minutes without any loss in sensitivity. This method can be applied to western blots using both chemiluminescent and fluorescent detection. This simple protocol allows researchers to better explore the analysis of protein expression in many samples.

An ultra-high-speed western blotting technique is developed by improving the kinetics of antigen-antibody binding through cyclic draining and replenishing (CDR) technology in conjunction with an immunoreaction enhancing agent.

A western blot (also known as an immunoblot) is a canonical method for biomedical research. It is commonly used to determine the relative size and ...

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase C&#946;/No receptor potential A (PLC&#946;/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Purification of Endogenous Drosophila Transient Receptor Potential Channels

Based on the assembling mechanism of the INAD protein complex, in this protocol, a modified affinity purification plus competition strategy was developed to purify the endogenous Drosophila TRP channel.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, ...