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- I used RIPA Buffer with Roche Protease Inhibitor. It was not on ice when I used it, but it was straight out of the fridge. The samples were allowed to lyse on ice at 4 degrees for 20 min before centrifuging and collecting the supernatant.
- Have not tried any protocol trouble shooting just yet, there is definitely sample loaded etc and transfer appeared to work as there is alot of binding at other MWs. Another blot that was run at the same time worked. I have considered running a lower concentration gel (to seperate the high MW proteins better) and transferring for a longer period to ensure every last bit of protein getstransferredto the membrane (at the cost of losing some lower MW proteins)
- yes, samples were denatured for 5 min at 95-100 degrees
- image attached
- yes, we use a protease inhibitor from roche. made up at 25x conc and diluted to 1x into the RIPA buffer immediately prior to the lysis of the cells.
Asked on Aug 14 2012
Thanks you for confirming the details.
The protocol looks fine to me so I am sorry we are unable to determine why the antibody not able to detect right size band? Perhaps you can try following steps for troubleshooting
- 70-80% cells confluency
- Heating lysates at 100C for 10 minutes or heating at 60-70C for 15-20 minutes could be tried.
- Try no primary control
- or finally as per your suggestion different gel can also be tried.
I hope these suggestions will help. Should this antibody fails again please let me know I will replace the antibody.
Answered on Aug 14 2012