Detection of Phosphorylated Neuronal Potassium-Chloride Cotransporter 2 via Western Blotting

Take a cell lysate containing potassium-chloride cotransporter 2, or KCC2 proteins, which regulate neuronal chloride levels.

Phosphorylation at regulatory sites disrupts KCC2 function, modulating neuronal activity. 

Add the lysate to beads conjugated with antibodies targeting phosphorylated KCC2. Incubate to allow binding, then wash to remove unbound proteins and cellular debris.

Treat with a denaturing buffer at a high temperature to release KCC2, then centrifuge to separate the beads.

Load the KCC2-containing supernatant onto an electrophoresis gel. Separate the proteins into bands to confirm the presence of phosphorylated KCC2.

Transfer the bands to a membrane, then incubate in a blocking solution to mask non-specific binding sites.

Incubate with primary antibodies targeting phosphorylated KCC2.

Wash, then incubate with enzyme-tagged secondary antibodies to label the primary antibodies.

Wash again, then add a chemiluminescent substrate, which reacts with the enzyme to produce light.

Measure the light intensity to quantify phosphorylated KCC2 in the lysate.

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 this step.

Next, mix 1 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 4 degrees Celsius, repeat two washes with PBS.

Carry out protein quantification on the cell lysates, and add 1 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 2 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 5 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 1 hour at room temperature using a blocking buffer. Incubate the membranes with appropriate dilutions of primary antibody and beta actin in blocking buffer for 1 hour at room temperature or overnight at 4 degrees Celsius. Then, give three washes of TBST for 5 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.