Hello,
Today I want to ask whether there is absolute way of
extracting proteins from cells for western blot.
I found out that there are numerous ways to extract proteins.
In my case, I treat ripa buffer to cells, making samples in tubes.
Then, I vortex them(I don't think the number of vortexing is not critical,
but i think too many times of vortexing should be avoided so it seems that i
ususally vortex the samples one or two times in general.)
After that, I put the samples in centrifuge.
After centrifugation(The conditions of centrifugation differ every time
but the differences are not significant in my view)
for about 10~15 minutes, I see the pellets in the bottoms of the samples and the proteins above. I extract the proteins.
I have had not troubles with the experiment results, but I found out that the ways of
extracting proteins differ according to people so I am concerned whether
my way described above is not the right way.
I want to get confirmed with my steps in spite of no troubles
with the research results and wonder whether I have to adopt only one, absolute way of extracting proteins from cells.
Thank you!
hello,
Just to add to your confidence i would say that you are using the correct method. just try and carry out the extractions on ice and maintain cool conditions to prevent any protein degradation.
as far as vortexing is concerned i do not find it very essential. we in our lab do not vortex the samples.
Hope it was helpful to you.
Best of luck!
Hi!
You do everything well. The reason of problem could be in different summary content of protein. RIPA buffer is OK. I use it too. Probably, you can increase time of lysis for dissolving of pellet. More, you should to measure protein content every time and put equal amount of protein in each WB well. Please, discuss with your colleagues how much protein they add into each well.
Regards,
Tatiana
I think the main factor in preparation is that the quality of protein decreases within few days. The faster you make gels from the lysate the better quality you will have. Adding cocktail inhibitors to your lysate buffer can maintain the quality of lysate for few weeks though. another factor is to keep the proteins dissolved because insoluble proteins will not separate on gel. To obtain higher amount of protein you can add lysis buffer to the tissue culture flask directly. Good luck!
Dear Aastha Dheer, Thank you for your answer and encouragement.
Good luck with your research and work, too!
Dear Tatiana Sukhanova,
Thank you for your answer. I was very delighted with your informative reply.
Good luck with your research!!
- Badges
- Science method