Cannot detect GFP using GFP antibody - (Feb/26/2010 )
ursae on Mon Jul 19 02:04:47 2010 said:
wad do u mean detect gfp alone?
Whr is ur protein localised? I had an experience in which my protein+gfp doesnt shown up in WB no matter what. Then it occurred that this protein is localised in the nucleus and my ripa buffer wasnt strong enough to lyse the nuclear membrane. After i switched to a strong lysis buffer, tada it showed up just fine.
Or other thing is that ur transfection didnt really work anyway.
Lastly, try to use another antibody from another company. ur ab might have spoilt/degraded. tat usually happens.
well, Ripa buffer is quite strong, and I guess your protein was not in all the nucleus, but in some insolube bodies (pml bodies...). Anyway if you do a SDS lysis and you cannot detect your protein something is wrong with the WB. Maybe the antibody. I would add a positive control and then you will know