To determine the concentrations of my protein extracts by the bradford method, I have to draw the standard curve, I use 10 different concentrations of BSA; 1 0, 20, 30.... 100µg/µL. Normally I would have increasing Do values, while the opposite, the values are decreasing. knowing that the BSA is diluted in the extraction buffer. I tried to take a protein extract to determine its Do. When I add a volume tothe Bradford buffer, I get a particular Do value. When I put a higher volume than the previous one of the same extract, the Do value decreases while it should increase. So where is the problem?
According to the method described by you, the Do value should increase depending on the concentrations (10 having the lowest and 100 having the highest reading). Certain basic things you should maintain. Try to take the readings from lower to higher concentrations against the blank (only extraction buffer). then, try to rinse the cuvette in the following higher conc. buffer before taking the reading. Try taking the conc. of BSA from 2mg/ml stock buffer.
Serial dilution is suggested to get the best pattern for your standard curve.. To confirm that, please use M1V1=M2V2 where the final volume , V2 are standardize for all the conc involved
As an example, u should prepare stock solution first (10mg in 10ml)= 1mg/ml BSA
Maybe take 5 ml of final concentration, so u should have:
blank: no extract (stock) + 5ml buffer
0.2 mg/ml: 1ml extract (stock) + 4 ml buffer
0.4 mg/ml: 2ml extract (stock) + 3 ml buffer
0.6 mg/ml: 3ml extract (stock) + 2 ml buffer
0.8 mg/ml: 4ml extract (stock) + 1 ml buffer
1.0 mg/ml: 5 ml extract (stock) +no buffer
Use the bradford reagent to all the conc and u can observe the pattern of the blue colour even before you run with the UV.
Best of luck!
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