Question

STANDARD CURVE PREPARATION

Always indicate the volume of water (or additional solution) to be added to complete any dilution.

a) A stock solution of 6mg/ml BSA is provided. Calculate the dilutions for the preparation of the

following BSA standards (2.0ml volumes of each): 60, 170, 500, 1500, 3000ug/ml.

Also include the dilution factor for each preparation.

On a separate sheet of paper with a heading of Calculations, include one complete sample calculation of this dilution calculation.

b) To 0.5ml of your unknown samples (and dilutions of your samples) and to 0.5ml of the standards add 2.5ml of Biuret reagent. Calculate the final concentration of BSA after addition of the Biuret reagent.

iv. neatly show one sample calculations.

c) Hypothetical absorbencies with respect to the concentrations in ‘a’ are: 0.16, 0.20, 0.28, 0.45 and 0.70.

v. Use computer software such as excel to plot a standard curve using these absorbencies against the concentration of each BSA standard prior to the addition of Biuret reagent (use concentrations as given in step 11a directly)

vi. Label the figure as Figure 1 and give it an appropriate descriptive title. The title should be above the figure.

vii. Plot a second standard curve using the same absorbencies (0.16, 0.20, 0.28, 0.45 and 0.70) against the concentration of the BSA standard after the addition of the Biuret reagent (as calculated in step 11b)

viii. Label this figure as Figure 2 and give it an appropriate descriptive title.

d) A sample prepared with 0.50ml of protein sample A and 2.5ml of Biuret reagent gave an absorbance of 0.51. Calculate the protein concentration of the original protein A sample, in g/ml, twice: once with each standard curve.

ix. On the calculation sheet (see iii), neatly show all the calculations required to solve for the concentration of the protein sample. Remember to perform this calculation twice: once with each standard curve. Include a title to separate this calculation from others on the page.

e) A protein sample was prepared with 5.0 x 10-2 ml protein sample B, 0.45ml water and 2.5ml of Biuret reagent . It had an absorbance of 0.22 in the spectrophotometer. With this data, calculate the protein concentration of the original protein B sample, in g/ml, using either standard curve.

x. On the calculation sheet (see iii), neatly show all the calculations required to solve for the concentration of the protein sample. Include a title to separate this calculation from others on the page. Refer to the Figure that was used to solve for the concentration.

Answer 1

Answer a)Stock concentration of BSA 6mg/ml

Now we have to dilute it to 60g/ml;170g/ml;500g/ml;1500g/ml;3000g/ml and Final volume of each sample is 2ml

So according to dilution equation

M1 X V1 = M2 X V2 put M1= BSA stock concentration converted to g i.e. 6000g/ml and M2 = all the given concentration and V2 = 2ml

Example 6000 X V1 = 60 X 2 = .02ml = 20l

6000 X V1 = 170 X 2 = .056ml = 56l

6000 X V1 = 500 X 2 = .166ml = 166l

6000 X V1 = 1500 X 2 = .5ml = 500l

6000 X V1 = 3000 X 2 = 1ml = 1000l

Dilution Factor (DF)

DF= Final volume/dilution volumes

1) 2000/20 = 100

2) 2000/56 = 35.71

3) 2000/166 =12.04

4) 2000/500 = 4

5) 2000/1000 = 1