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

Abstract