I am lysing my cells directly in Laemmli buffer and have to quantitate protein concentration for loading in SDS-PAGE wells .How can I quantitate the protein ?
And I also want to detect a membrane bound protein. What should be the proper amount of protein to be loaded in wells to be detected by western blot?
I think in Laemmli buffer you can't quantitate your protein. But you could load different amounts of protein to the gel (e.g. 1 ul, 5 ul, 10 ul, 15 ul, etc) so after staining you will see how dense youre protein is and on western blot you will see what amount is enough in the future.
For membrane proteins a simple Laemmli treatment couldn't be enough. Maybe you have to degrade the membrane to get your membrane-bound protein by sonication, for example.
@ wassim..but in literature i have found that detergents interfere with folin lowry method,
You can precipitate your protein and quantify using Amido Black. See the protocol at http://www.rz.uni-karlsruhe.de/~db45/Methods/Protein/Amido_Black.html or you can use TCA precipitation, as given in the Amido Black protocol at Nature Signaling, http://www.signaling-gateway.org/data/cgi-bin/Protocols.cgi?cat=0 (scroll down to the bottom of the page).
thanks for the answer @ Daniel, but i just want to know whether BCA method can be reliably used to quantitate proteins in lammeli buffer and can RIPA buffer be used to extract protein and quantify with BCA and lowry methods
No, ionic detergents are not generally compatible with BCA or Lowry. Most RIPA formulation use SDS, so instead you need to consider different methods for protein concentration estimation in your extracts. A280 absorbance measurements are not good estimates for total protein, since nucleic acids can contribute to this signal, and different proteins have widely different extinction coefficients.
in order to normalize protein amount in laemmli buffer, you should prepare a first coomassie blue gel, quantify band by image J and after you should run your western blot
BCA is compatible with ionic detergents to a certain concentration, ie SDS 5%. However, in your Laemali buffer, your SDS concentration is 10%. Therefore, precipiation with TCA and quantification with amidoblak seems to be the best solution.
The BCA Assay is compatible with Laemmli buffer with regards to SDS (up to 5 %). Most Laemmli buffer protocols use even less SDS than 5%. However, your bromophenol blue (598 nm max absorbance) will interfere with BCA (562 nm). Nevertheless, run a Laemmli blank with your sample dilution along your sample in the assay and you will get a good estimate of your protein concentration (not as good as without bromophenol blue, but still good). I tested it by myself using BSA in 1X Laemmli buffer of different concentrations in different dilutions. Concentration was just of by a margin of ~10% from true concentration with a 1:5 dilution. Western Blot normalization will account for residual differences in protein amount anyway.
I also think staining your gel is a good option as long as you have enough sample material to do that.
may be, you can run gel, stain with coomassie blue and quantification by image j !
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