I am planning to seed 2x10e5 cells per well in a 6 well plate. How much ripa buffer i need to add ? What will be the amount of protinase and phosphatase k inbitor cocktail mix to add with ripa buffer?
Hello Md Abu Siddique
The recommended ratio is 1x10^6 cells in 100 ul RIPA buffer. For adherent cells you may add RIPA buffer in each well placed on ice. Scrape adherent cells off the well using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube. Add the proteinase and phosphatase inhibitor cocktail fresh to the lysis buffer at the time of lysis.
You need to add 100-150 ul RIPA buffer in each well of a 6-well plate. So, if you add 100ul RIPA buffer per well, you will need 0.6 ml of RIPA buffer for 6 wells. Make a little extra about 0.7ml RIPA buffer.
The proteinase and phosphatase inhibitor cocktail comes as a 100X solution. You need to add 1X final concentration to the RIPA buffer. So, add 7ul of proteinase and phosphatase inhibitor cocktail (100X solution) to 693ul of RIPA buffer. This will give you 0.7 ml RIPA buffer with 1X concentration of proteinase and phosphatase inhibitor cocktail.
Best.
Malcolm Nobre Thank you a lot for your answer. I am planning to seed 2e105 cells per well. That means 40ul RIPA buffer per well? Moreover if i add only protinease K inhibitor how much i need to add with ripa buffer ?
Hello Md Abu Siddique
Yes, 40ul RIPA buffer per well.
Proteinase K inhibitor like PMSF may be used. You should add 0.1-1 mM in the final volume of your RIPA lysis buffer.
Prepare 100mM PMSF by adding 17.4 mg of PMSF per ml of isopropanol. Dilute this stock (1:10) to obtain 10mM PMSF (diluted stock).
So, if you add 1mM PMSF in lysis buffer, you will have to add 4ul of 10mM diluted stock of PMSF in 36ul of RIPA buffer to get 1mM PMSF (proteinase K inhibitor) in 40ul of RIPA buffer.
Malcolm Nobre Thanks a lot for your answer. I am using https://www.sigmaaldrich.com/US/en/product/sigma/ppc1010 .
This protease and phosphatase inhibitor. But i am not getting how muh i need to add with the RIPA buffer.
@Malcolm Nobre...Hello Sir.
Sir, I want to isolate protein from PBMCs using RIPA buffer with proteinase inhibitor cocktail. Before going for protein isolation, I did Cell counting. Sample size is 100. So, for every sample cell count is different. It's mentioned in many protocols that for 1x 10⁶ cells , use 100ul RIPA buffer. So, sir how much ripa should I add for different cell count like for one sample the cell count is less that's 0.9x 10⁵ cells. So how do I know how much RIPA to add.
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