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.
