I am working on protein isolation by using RIPA buffer, the composition which I am using is (50 mM TrisHCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA), the composition which I have mentioned is it 10X? Do I need to dilute this stock concentration into 1X before lysing the tissue?
Najmin Ahmed
The composition you have mentioned is ready to use!
It's 1X concentration. You need to add 0.3 to 0.5 ml of RIPA for 3-5 million cells or use 300uL per 5mg of tissue.
NOTE: RIPA or any lysis reagent should be used at a temperature below 4 C to avoid denaturation of protein. Perform all your experiments with ICE-COLD buffer.
Cheers
Hello Najmin
In addition to the above, protease and phosphatase inhibitors can be added just before use to RIPA buffer depending on your requirement. This will help to prevent proteolysis and maintain phosphorylation of proteins.
Best Wishes.
Hi Najmin, though it is the standard composition of RIPA buffer no doubt , but it is often unlikely that you have received it as 1x, usually it is provided as concentrated stock like 5x, 10x, 20x etc and you must need to dilute it, unless it is prepared by someone in the lab in your vicinity. It is better to check the fact sheet of this RIPA provider, because it is even not economical at all that anyone uses 1x RIPA from any company, as it is an expensive choice unless you have less work load. I know that still companies are providing it as 1X ready-to-use, but in this case too you must cross check from the manufacturer's page, and just reading out its composition from the bottle you cannot declare that whether it is 1x or concentrated one.
Regards....
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