Hi everyone,I have a purified protein sample how can I measure the this purified protein concentration to load the sds-page?
There are several commonly used protein assay methods, including Bradford, Lowry, and BCA. Reagents and kits for all of these are commercially available. A spectrophotometer or absorbance plate reader is required.
There are several commonly used protein assay methods, including Bradford, Lowry, and BCA. Reagents and kits for all of these are commercially available. A spectrophotometer or absorbance plate reader is required.
You may run the gel with a known marker protein in the SDS PAGE and then analyze your relative band density to make a plot. I'm not sure how accurate such band analysis could be done.
There are a number of protein quantification assays available like the BCA or Bradford assays which vary in the level of accuracy obtained once you use them. However, you can alternatively use" gel approximatation" using by loading a specific protein ladder with known band sizes and concentration..Then you compare the size of the sample band to the size of the protein ladder in order to approximate the quality of protein in your sample
The simple way to spot parallel-serial two-three fold dilutions of your sample protein and a protein with the known concentration (let say, BSA) onto nitrocellulose membrane and stain it with 5 times diluted Coomassie gel stain. The accuracy is quite enough for your task, I think.
If you have pure protein and know the sequence you can check concentration using A280nm.
Expasy Protoparam calculates the concentration for you from sequence. Use the buffer that the protein is in as blank. You can also run a sample in 6M GnHCl to unfold the protein and ensure that all tryptophans etc are exposed and compare to unfolded sample. If you have a nanopsec then volume of sample is not an issue.
Bradford/BCA as suggested above are also easy to use
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