How to quantify recombinant expressed protein if some is secreted to medium and some gets attached to the membrane? (also some inside the bacteria but I'm not interested in this fraction).
I quantified the protein secreted to medium using PEG precipitation and BCA kit but this method cannot be used with the protein left attached to the membrane because has interference with lipids. What could you recommend me to quantify this portion of expressed protein?
Catalina,
You could try the CBQCA Protein Quantitation Kit from Life Sciences. It is about USD$200 and is a very sensitive assay for quantitating proteins in solution. Alternatively they also have NanoOrange protein quantitation reagent (N-6666). However, CBQCA is better for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.
I am attaching two links to a couple of articles that might be useful to you.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC217593/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246389/
Que tengas un buen dia !
Bradford assay I find is easy and yeah BCA is as well good....but do you intend to go for specific protein estimation, for that one should always run ELISA and co-relate with BCA or Bradford.
Boss use bradford method or NanoDrop if avilable, Bioanalyzer also gives accurate results
The assay is important. But, first, you must ensure that most of the recombinant protein is secreted. So, the time course, transformation conditions and the entire experimental design must be optimized to ensure maximum secretion. Can the secreted protein get degraded in the media..(serum free?)?? Perhaps you can check if others use protease inhibitors in their protocols. Hope this helps!
You can use a detergent such as Triton X-100 0.5-1% (w/v) in order to solubilize membrane proteins, then quantify proteins. Also, you can use a protocol to obtain the membrane protein fraction.
Hello Everyone!!
Thank you for all your answers!! and your help.
I guess BCA and Bradford quantifies total protein but I need to go for specific protein quantification (just my recombinant protein expressed). I do agree I would have to go with ELISA as Mustafa recomended it but I don´t have a protocol for that yet.
I would try to develop it and co-relate de data with BCA/CBQCA and Bradford and get the membrane protein fraction using a detergent.
I´ll let you know how it goes. Thank al!!
what kind of protein you are targeting? It will be help in developing a protocol for ELISA
Yes, If your expressed protein has tag, you can purify it using affinity chromatography to have your secreted and membrane (using mild detergent extraction) bound protein. This will eliminate your cumbersome protocols to quantify the protein of interest among other proteins in media/cell lysate
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