I am purifying a recombinant protein (expressed as inclusion bodies) on a ion exchange column eluting with a gradient of NaCl. A sharp peak is seen in elution which scores pretty high on absorbance at 280 nm, However there is no or sometimes only a very faint protein band seen on SDS-PAGE. Would anyone know why the band intensity on PAGE does not correspond to absorbance at 280 nm?
Though the literature says that till 1M NaCl there is no interference. But I have myself witnessed that even at 0.6 to 0.8M NaCl in 50mM Tris-HCL also interferes reading at 280 nm. If you really want to check you desalt your protein by concentrator and repeatedly wash with water or buffer and then recheck your protein concentration at 280 nm. There will be a huge difference...
First, it may depend on the pI of the protein, Coomassie stain tends to bind less to acidic proteins due to its negative charge, resulting in faint bands. If your protein has higher molecular band in SDS-PAGE than the theoretical MW, this can be the case as well.
Secondly, you can have low mw contaminants not showing up in the gel, or a low concentration of high-aromatic content proteins (any MW, but too faint to be seen on gel), which ultimately account for your total A280.
HTH
Marco
I think you should dilute or desalt your samples and estimate protein concentration using proper protein assay method (good selection you may find here: http://www.piercenet.com/guide/protein-assay-selection-guide). Personally I like home made microbradford assay (if you want detailed protocol, please contact me), but it requires detergent removal. Some proteins do not absorbe at 280 due to low or non aromatic amino acids content. For such proteins also Bradford method won't work well and better to use peptide bond specific assays (Lowry, BCA for example).
As Marco states, you may also have low MW compounds absorbing well at 280 and that produce your sharp pik. There is a good formula to follow for spectrophotometric determination of protein concentration C(mg/ml)=(Absorbancy at 235-Absorbancy at 280)/2.51 (according to: Whitheker and Granum 1980, http://www.sciencedirect.com/science/article/pii/000326978090024X).
This formula can help you to find out if your high 280 pik is in fact protein or not. If not, it won't have peptide bonds and won't absorb at 235 nm and you will get negative result. In my hand this formula is very accurate for protein concentration determination if there is no color or organic contaminants in samples.
In case you do have protein but it is not stained by regular methods in gel, buy silver staining kit (or make it yourself) and stain protein in gel with silver. Regular coomassie stains won't stain proteins with low content of aromatic amino acids
Hope this helps..
You only talk about absorbance at 280 nm, but did you check the spectra of your sample (say between 220 and 350 nm) ?
This could be a good control to test if your spectra looks like the one of pure protein, or if there are some other compounds present in your sample. In fact, lots of other molecules can absorb at 280 nm, being able to contribute to your measured absorbance, which can explain why you only see a small amount of protein on your gels.
Hope this will help you.
Thank you everyone for detailed responses. It gives a lead into identifying the problem.
Request to Krzysztof Treder : would you please send your micro Bradford protocol?
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