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[dcch]

Hello. I am new to total protein extraction from mammalian cell culture. I plan to use RIPA buffer without sodium deoxycholate for the extraction (sodium deoxycholate is not available in my lab ) However, when i searched through the web for RIPA buffer protocol, basically 3 versions of Tris composition in RIPA buffer were present, which is 25mM Tris-HCl pH7.6, 50mM Tris-HCl pH7.4 and 50 mM Tris (I think it is Tris-base?) pH8.0.

Which type of Tris, its concentration and pH should i follow?
Can i substitute NP-40 with Triton X-100? I do not have NP-40 here...
In a modified RIPA buffer, EDTA is suggested to add at 1mM. Will it be ok?

For the protocol to perform total protein extraction, some of them suggested to put the RIPA buffer into the well, agitate for 5 minutes in cold, scrape and spin down; while some suggested aggitate for 15 minutes. Which one is the most suitable for mammalian cell culture (like HeLa, HepG2 etc)? After that, should sonication be performed again? Lastly, will spin down the cell lysate at 13k rpm for 5 minutes sufficient to seperate cell lysate and protein?

Or...can some body kindly share your protocol with me?
Thank you...I am quite new in protein experiment

[selenocysteine]

Hi, how is your progress with this problem?

dcch, on Sep 17 2009, 11:14 PM, said:

Which type of Tris, its concentration and pH should i follow?
Can i substitute NP-40 with Triton X-100? I do not have NP-40 here...
In a modified RIPA buffer, EDTA is suggested to add at 1mM. Will it be ok?

Ultimately it will depend on trial and error. Are there other labs that work on the same protein as your and do the same assays? See what they use in their published research. I plan on using TrisHCl pH 8.0 without EDTA in my RIPA buffer. There is also a phosphote buffered version of RIPA. NP-40 is the trademarked name of a detergent that is no longer manufactured. Look for Nonidet P-40 substitute (from Fluka, I think) or Igepal CA-630 from Sigma.

Quote

For the protocol to perform total protein extraction, some of them suggested to put the RIPA buffer into the well, agitate for 5 minutes in cold, scrape and spin down; while some suggested aggitate for 15 minutes. Which one is the most suitable for mammalian cell culture (like HeLa, HepG2 etc)? After that, should sonication be performed again? Lastly, will spin down the cell lysate at 13k rpm for 5 minutes sufficient to seperate cell lysate and protein?

Or...can some body kindly share your protocol with me?
Thank you...I am quite new in protein experiment

Either method could work. You may have to optimize for your protein of interest. Is your protein cytoplasmic or nuclear (or unknown)? If it's cytoplasmic you might not need to sonicate...I also read once that RIPA buffer can disrupt nuclei...I don't know if that's correct, though. To prepare cytoplasmic extract I centrifuge my samples at 17,000 x g for 10 minutes at 4 degrees C and use the supernatant.

This website is helpful:
http://pingu.salk.edu/~sefton/Hyper_protoc...munoprecip.html

Edited by selenocysteine, 24 October 2009 - 12:43 AM.