Suppose that you had run out of Coomassie blue reagent to stain protein in the SDS
-PAGE gel, but still needed to determine how much kinesin - GFP you had in your purified sample. You do, however, have a sample of GFP - tubulin of known concentration, and spectrophotometer available. How could you determine the concentration of kinesin - GFP using the GFP - tubulin and the spectrophotometer? Remember, GFP is green no matter what other protein it is attached to.
If you have a spectrophotometer, you can use A280 to estimate the amount of total protein in your sample versus a known standard such as BSA or IgG. You can also use the Bradford assay or a similar assay if you have the reagents available. Fluorescence intensity fluctuates - even if your spectrophotometer had the ability to excite and measure the emission from GFP, it would not be as reliable a measurement as the other methods. Of course, the disadvantage versus using a gel is that you have to make the assumption that your sample is pure.
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