Biuret and Bradford Assays of the Proteins Isolated from Mil

Biuret and Bradford Assays of the Proteins Isolated from Milk and Eggs BIOCHEMISTRY 154 edure Prepare 10 mL of a 1 % solution in TES buffer (0.05 M Tris-HCl, 0.005 M EDTA, 0.05 M NaCI) for each of your isolated protein samples from egg (that is, 0.1g in 10 ml TES). Casein has low solubility in water, so we will not use it for this experiment. You will determine the amount of protein in your isolated sample by doing a Biuret proteirn assay. BSA (bovine serum albumin) will be used as the standard. The Stock BSA solution is 3 mg/ml in TES buffer. Set up 10 test tubes as shown below. Tube 1 is the blank. Tubes 7 and 8 are your two isolated protein samples from egg. Tube # td Prot Biuret Average Subtract 12 2.0 200 0 0 0.20.2 0.4 0.4 0.8 0.8 1.2 3,0 30 3.013.013.013.013.013, 3.0 3.013.0-3.0 0 3,0 3,0- 3,0 le 013 |.044 | Blank 0.0 I8 4IO Tube# 1 Protein Conc. 3. Add 3.0 mL of Biuret reagent to each tube and mix. Let the tubes stand at room temperature for 30 minutes, then measure the absorbance at 540, zeroing the instrument on the blank. 4. C onstruct a standard curve by plotting absorbance versus concentration, and determine the concentration of your protein solution. Do not include absorbance values greater than 1.0 to draw the \"best fit\" line. 3. If your protein samples have absorbance greater than 1.0 you should repeat the Biuret assay by using less protein Calculate the mg of protein per 10 mL of isolated sample. Calculate the % yield (that is, for the isolated protein, relative to its dry weight) radford Assay. u will also assay the proteins using Bradford method. Make 1:50 dilution of the standard tein (provided) and also make 1:50 dilution of the solutions of your isolated proteins. Repea tthot ( OU will us the diluted protein solutions, (b) you will use Bradford

Solution

BSA initial concentration 0.1 mg/10ml

0.1x0.2= 0.02

For tube 3,

0.1*0.4= 0.04

Similarly for egg protein,

The initial concentration was 3mg/ml

3*0.5= 1.5

3*2= 6