Today in lab we created a standard to measure protein concentration based on the absorbance of different concentrations of the proteins Bovine Serumm Albumin (BSA) and Bovine Gamma Globulin (BGG, technically known as IgG, for Immunoglobulin G) in 1x Bradford dye reagent, or more precisely, an acidic solution of Coomassie® Brilliant Blue G-250 dye, which absorbance maximum shifts from 465 nm to 595 nm when binding to protein occurs.
We added a range of protein concentrations to the dye reagent, and measured absorbance at 595 nm. The more protein there is in a sample the more dye will be shifting absorbance to the wavelengt we used and the more absorbance will be observed. That is the principle of the Bradford method.
Using such standards (BSA abd IgG) we are going to get an idea of the concentration of our problem sanple, GFP. Ideally, we would use a range of GFP concentrations to create a standard and make an accurate measurement, but let's pretend we just discovered a new protein, and we know nothing about it. We use known standards to get a rough idea of how much protein we purified.
Tomorrow we will run our GFP sample in a native polyacrylamide gel, measure the distance the band migrates in the gel, compare it with some protein standards, an estimate the molecular weight (size) of the GFP. Then we'll have an estimate of how much "novel" protein we purified, and what size it is.
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