Storing protein extracted from tissues for Western blot - (Feb/19/2008 )

Hi,

I have difficulty getting good results for my Western blot and found out from months of trouble shooting that the signal of my protein actually deteriorates after storing at -80 Deg C for a few weeks... even a few days. Somehow, even after transfering to NC membrane, the protein signal deteriorates (I store my post transfer NC membrane dry at room temperature in between two whatmann 3MM paper). The actin signal is still strong ... just the receptor protein that I am interested in seemed to run down.

Can anyone help with methods of storing my protein samples or even my NC membrane. Currently, my samples are extracted from brain tissue in Tris-EDTA buffer with protease inhibitors added. I then add in 2X Sample buffer and boil at 95 Dec C for 5min and store in small aliquotes at -80DegC.

Help!!

Sindy

Out of curiosity, have you compared new lysates with old lysates to compare expression? Just wanted to know if the antibody is still working fine.

Hi Malik

That was how I ended up having to do my protein extractions, gel, transfer and process the Western blot all in one day... did a time line on my protein lysates... tried ones extracted fresh and those stored at -80DegC for 1week, 2 week, 1months etc and found a reduction in signal. I am much puzzle as to how this can happen... how do you process and store your protein sample for Western blot? I also tried storing my NC membrane dry for a week or so and compared that with freshly transferred membrane..and found reduction in signal...

Hi SKLL,

we normally extract and store all samples in -80C. Sometimes we continue to western on the same day up to transfer and continue the next day.

It is weird what is happening with your samples. If your samples are degrading in -80C, may be you should store them in liquid nitrogen.

Hi Malik

Yeah... the protein I am working with is kinda "weird".... we haven't got liquid nitrogen facility in our lab.. so, theo nly way around is to run the sample immediately post extraction and continue straight on. I was wondering how you store your post transfer membrane... just met someone with Western blot background and he store his membrane in blocking buffer (5% skimmed milk) overnight...

Sindy

Hi Malik

Thanks. I would assume then that there is no such thing as "overblocking"... do you need to leave it with gentle aggitation....
Just need to see if I can continue the next day as I need to collect my kids from school... since I can't store my protein samples... probably the best way is to store the membrane.
Sindy

hi Sindy,

gentle agitation is always good. You could leave the membrane in primary antibody (with agitation) for 2-3 days before continuing with secondary.

i did noticed degradations of proteins quantity on my total extracts. Activity was also reduced that's why i went to cytoplasmic/nuclear extract preparation (not sure if it's relevant to your situation but i share my exp) and my extract have a final glycerol concentration of 30%. Iafter extraction i put my samples aliquoted in liquid nitrogen and store at -80°. Activity of the nuclear extracts was ok for weeks, and quantity is stable over months.

HI Fred and Malik

Thanks... tried out storing my post-transfer membrane in blocking buffer ON at 4DegC and it seemed to work! Also, am testing storing my protein extract ..in sample buffer (final concentration has 25% glycerol), snap frozen in Lq Nitrogen and then store at -80DegC... will get results in a few hours!

Thanks
Sindy