3 replies to this topic

[Garp]

Hi there,
I´m trying to coIP two proteins after cotransfection of their corresponding vectors.
One Protein should be around 200kDa and is Flag tagged, the other one´s ~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 don´t 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 don´t IP the same day).
Thanks in advance!

[rajgene]

Garp, on Jul 1 2009, 03:12 AM, said:

Hi there,
I´m trying to coIP two proteins after cotransfection of their corresponding vectors.
One Protein should be around 200kDa and is Flag tagged, the other one´s ~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 don´t 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 don´t 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.

[Garp]

the buffer is supplied with 1x protease inhibitor cocktail. I was just wondering wether  the needle could do my proteins any harm....

[rajgene]

Garp, on Jul 1 2009, 05:55 AM, said:

the buffer is supplied with 1x protease inhibitor cocktail. I was just wondering wether  the needle could do my proteins any harm....

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

Edited by rajgene, 01 July 2009 - 06:37 AM.