many bands of smaller size than expected - (Jul/01/2009 )
Hi there,
Iīm trying to coIP two proteins after cotransfection of their corresponding vectors.
One Protein should be around 200kDa and is Flag tagged, the other oneīs ~50kDA and GFP tagged. After blotting I get a lot of smaller sized (around 10 bands of sizes from 40-100 kDa) fragments with the anti Flag (M2) AB and only a very weak band of the correct size. Detection of the GFP construct works nicely and in the non-Flag transfected control I donīt see any bands with the Flag AB, so the smaller bands seem to be specific.
My question is, wether I could have fragmented my bigger protein during lysis....
Lysis conditions: cells are trypsinized, centrifugated and taken up in RIPA buffer --> 10min 4°--> shearing of DNA by vigorously passing trough a needle 10X -->cf.
Does freezing affect Protein integrity (I usually donīt IP the same day).
Thanks in advance!
Garp on Jul 1 2009, 03:12 AM said:
Iīm trying to coIP two proteins after cotransfection of their corresponding vectors.
One Protein should be around 200kDa and is Flag tagged, the other oneīs ~50kDA and GFP tagged. After blotting I get a lot of smaller sized (around 10 bands of sizes from 40-100 kDa) fragments with the anti Flag (M2) AB and only a very weak band of the correct size. Detection of the GFP construct works nicely and in the non-Flag transfected control I donīt see any bands with the Flag AB, so the smaller bands seem to be specific.
My question is, wether I could have fragmented my bigger protein during lysis....
Lysis conditions: cells are trypsinized, centrifugated and taken up in RIPA buffer --> 10min 4°--> shearing of DNA by vigorously passing trough a needle 10X -->cf.
Does freezing affect Protein integrity (I usually donīt IP the same day).
Thanks in advance!
is your buffer has sufficient amount of protease and phosphatase inhibitors? if not use atleast protease inhibitor tablets from ROCHE.
the buffer is supplied with 1x protease inhibitor cocktail. I was just wondering wether the needle could do my proteins any harm....
Garp on Jul 1 2009, 05:55 AM said:
hmm that could be a reason. usually hardcore lysis (like sonication) are not recommended to coip experiments but don't know how harsh is your syringe lysis. i was interested in nuclear proteins coip but could not achieve it in home made lysis buffer so i switched to M-PER lysis buffer from piercenet/thermoscientific..it works great without syringe or sonication or freez thaw lysis.
ALSO
the protease inhibitors have limited efficacy at 4°c in the buffer, hence its better to use tablets and make fresh buffer before critical experiments like coip.
hope this helps