SUMOylation Assay: An In Vitro Technique to Detect the SUMOylation Status of Substrate Proteins by Immunoblotting
Transcrição
Add 3 microliters of 100 nanograms per microliter of purified tag-K-bZIP in the de vitro SUMOylation master mix reaction. Then, mix the contents of the master mix gently, and incubate at 30 degrees Celsius for 3 hours.
Next, add 20 microliters of 2x SDS-PAGE with loading buffer to stop the reaction. Then, heat the samples at 95 degrees Celsius for 5 minutes to denature the protein.
Next, load 20 microliters of the sample on a 10% SDS-PAGE gel, and run the gel at 80 volts for about 120 minutes till the dye reaches the bottom of the gel.
Once the electrophoresis is over, disconnect the gel apparatus, and transfer the gel in semi-dry transfer buffer for 5 minutes. Then, soak the PVDF membrane in another container filled with methanol for 1 minute.
Remove the PVDF membrane from the methanol, and soak in semi-dry transfer buffer. Then, gently agitate the membrane for 5 minutes.
Remove the safety cover of the semi-dry electrophoretic apparatus. Pre-wet a filter paper, and prepare a gel sandwich on the bottom of the platinum anode. After securing the cathode plate in the safety cover, run the blot at 15 volts constant current for 90 minutes. Then, turn the power supply off and take the PVDF membrane out, after disconnecting the semi-dry apparatus.
Now, block the PVDF membrane with blocking buffer, for 1 hour at room temperature on an orbital shaker at 30 revolutions per minute. Next, hybridize the PVDF membrane with anti-p53 antibody and blocking buffer for 12 to 16 hours at 4 degrees Celsius, on a suspension mixer at 30 revolutions per minute.
Then, take the PVDF membrane out, and transfer in a container filled with TRIS-buffered saline with Tween-20. Next, soak the PVDF membrane in TRIS-buffered saline with Tween-20 for 30 minutes on the orbital shaker at 45 revolutions per minute. Then, hybridize the PVDF membrane with anti-rabbit antibody conjugated with horseradish peroxidase diluted in blocking buffer, for 1 hour at room temperature on the suspension mixer at 30 revolutions per minute.
Next, wash the PVDF membrane with TRIS-buffered saline with Tween-20 thrice. After washing, soak the PVDF membrane in TRIS-buffered saline Tween-20 for 30 minutes on the suspension mixer at 45 revolutions per minute. Then, replace the TRIS-buffered saline Tween-20 with phosphate-buffered saline to preserve the PVDF membrane at 4 degrees Celsius for up to 12 hours.
Next, mix the enhanced chemiluminescent substrate reagents 1 and 2 in a 1:1 ratio. Then, remove the PVDF membrane from the phosphate-buffered saline, and blot briefly with punch pockets to absorb the excess moisture.
Immediately, add 400 microliters of the chemiluminescent reagent to the membrane surface, and wait for 3 to 5 minutes. Briefly blot to drain the excess chemiluminescent reagent, preventing the membrane from drying completely. Use luminescence imaging system to expose the blot.
