Bradford assay - (Jan/09/2008 )
Hi, I am facing a problem with bradford assay. First of all, it is tough to get the desired curve. Secondly, I have been taught to make the graph on MS Excel. The program itself gives the y=mx+c equation, but I am not clear how it works. I understand that the principle is quantifiation of proteins, so i get the primary curve with BSA and ultra pure water. Then, when I repeat with unknown protein and water, I am finding it difficult to get the calculation part.. Can you please help?
-anujd-
QUOTE (anujd @ Jan 10 2008, 12:23 AM)
Hi, I am facing a problem with bradford assay. First of all, it is tough to get the desired curve. Secondly, I have been taught to make the graph on MS Excel. The program itself gives the y=mx+c equation, but I am not clear how it works. I understand that the principle is quantifiation of proteins, so i get the primary curve with BSA and ultra pure water. Then, when I repeat with unknown protein and water, I am finding it difficult to get the calculation part.. Can you please help?
How many standards (BSA) are you using and what concentration? Are you diluting your BSA in the same stuff your protein is in? I always do a couple dilutions of my samples (in triplicate). Then I use the absorbance(s) that are in the range of my BSA samples.
-Clare-
QUOTE (anujd @ Jan 9 2008, 05:23 PM)
Hi, I am facing a problem with bradford assay. First of all, it is tough to get the desired curve. Secondly, I have been taught to make the graph on MS Excel. The program itself gives the y=mx+c equation, but I am not clear how it works. I understand that the principle is quantifiation of proteins, so i get the primary curve with BSA and ultra pure water. Then, when I repeat with unknown protein and water, I am finding it difficult to get the calculation part.. Can you please help?
you need the regression for calibration; I think, although ticking the box for a regression through the point zero, it is not in any case at zero (X = 0, Y = 0) in Excel; better use more scientific orientad programs such as Sigmaplot...
-The Bearer-