I am new to western blot and experienced that when i store my lysate at -20, and freeze thaw it , and run the gel, it works wel and gud band seen after developmentl, but after that if i repeat the same sample again after freeze thawing, no band was seen after development. I have read that protein lysate shud be stored at -20 or -80 and in aliquotes.
So, can by storing lysate at -80 and in aliquotes, if i run the sample sample and can get gud band.
I have not made aliquotes as i my lysate was suspended in very less buffer and also as i thought repeated freezing thawing of same lysate will not have problem if i am freeze-thawing it just two/three times.
can anybody can tell me how i can get gud band everytime by same sample once i freeze-thaw it.
thanks in advance.
use enzyme inaktivation for extraction and storage! for western store probes in the appropriate buffer system (such as lämmli)! aliquote your probes! Nice day
I agree with the comments above and wanted to add that you should store your aliquote of the sample in SDS loading buffer (Laemmli buffer) in the amount you would need for a new gel. Before freezing just boil the sample at 95C for 5 min and then store at -20C. The protein is then already denatured and should be stable at least for one freezing step in the buffer also containg protease inhibitors resp. phosphatase inhibitors or other inhibitors. Good luck!
i am extracting the protein by boiling with 2x sample buffer (laemilli buffer) only. so my protein is actually stored in that only. my sample is working for only one time , i mean only for one freeze thaw. Again if i run the gel after freezw-thawing the same aliquot it doesnot work. So i just wanna know if just by one-freeze thaw cycle, protein get degrdes, i thought atleast 2-3 times freeze -thaw cycle, it will work.
I was just wondering since the this sample buffer does not contain protease inhibitor as we put in RIPA lysis buffer.so do protein are more stable in RIPA buffer as they contain protease inhibitors, but i also read that the SDS in sample buffer denatures the proteases so there is no need of protease inhibitors in sample buffer.
For protein stability, Is RIPA buffer is better?
Hi Pallavi, from my experiences, I would say you chose the correct way, because boiling in SDS sample buffer (Laemmli) is better than RIPA buffer with protease inhibitors, but it really depends on the proteins you are looking for. Did you tried in your second gel run to develop classical loading proteins like GAPDH, beta-actin, beta-tubulin? Those should at least work as a kind of a positive control otherwise I am wondering which composition of Laemmli buffer you are using.
2x laemmli buffer should be blue not yellow, please check your pH. I had the problem with the color in former times as well.
Never store protein in Laemmli buffer at 4 degrees, this will be totally unstable.
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