Hello everyone,
I lyse the cell culture (THP-1) with RIPA buffer but it interfere both the nano drop and bradford assay. Can I only use the water to lyse the cell?
Thank you.
Nanodrop not for cell lysate! you have to use any colorimetric assay/available kits that can tolerate RIPA.
If water can lyse the cells we wouldnt be alive, isnt it? :)
My suggestion is try RIPA, load equally on the gel and use a loading control staining (Tubulin for example) for your blot.
It is very essential to have a detergent in your lysis buffer. Water alone does not work efficiently!
Check out this:
http://bitesizebio.com/9107/how-to-lyse-cells-for-protein-extraction/
There's several buffers available, just check any of the papers. I use a HEPES lysis buffer which shouldn't interefere with Bradford: 250mM NaCl, 0.5% NP40 (or IGEPAL) and 20mM HEPES pH 7.5. Good luck!
using water only will not lead to a possible solution and that will not result in lysis. You can try using Triton X-100, (in dilutions) for lysis of your cells. usually it does not interfere with the proteins.
For example, 0.1-0.5% TritonX-100, diluted in PBS WORKS REALLY GOOD.
In addition to BCA assay, you can use the BioRad Detergent-Compatible kit. It works very well as an alternative to Bradford in instances where detergents are present.
http://www.bio-rad.com/en-us/product/dc-protein-assay
I agree RIPA interfere with nanodrop. Once i had a problem of interference of RIPA with Bradford and then i found that the RIPA i was using was without EDTA. I tried RIPA with EDTA and from that date its working for me.
Hi Wiyada, cell lysis with Water will work for cells without a cell wall i.e. mammalian cells. I commonly use dH2O with 0.1% Triton-X, I then do a freeze thaw cycle on the lysate to ensure thorough lysis. The Triton should not interfere with your protein assay at this conc. (I use the BCA assay for protein determination)
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If the protein estimation is the problem just check if you're making you're standard proteins in the same lysis buffer you used to extract protein (e.g. RIPA) - doing this should give you a more accurate slope when you're doing the linear regression in standard curve
Hi, I use the BCA assay for protein estimation for the western blot as RIPA buffer does not interfere with the analysis. Water should work as well, but to how efficiently I can not say.
Also for the BCA assay, I get very nice results when I dilute by at least 1/10.
This is the buffer that I use to lyse:
http://www.ncbi.nlm.nih.gov/pubmed/19737230
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