4. A Bradford assay was conducted to determine the total protein concentration i

Question

4. A Bradford assay was conducted to determine the total protein concentration in a sample. 2 uL of the original sample was diluted to 100 uL with buffer before performing the assay. The dilute sample gave an absorbance at 595 nm of 0.225 Using the data for the standards below, determine the total protein concentration in the original sample [protein] (Hg/mL) A595 0.7 0.6 0.5 y = 0.0004x 0.01 1 7 25 125 0.087 2500.113 5000.197 750 0 1000 0.429 1500 0.608 0.008 0.3 0.2 0.1 0.295 500 1000 1500 2000 [protein] (ug/mL)

Explanation / Answer

4. From the graph, by drawing perpendicular on the line it is found that the concentration of protein was 560 µg/ml

As the dilution factor is 50 ( 2 µl to 100 µl) the amount of protein will also be also increased by 50.

So the concentration of protein in the original solution will be = 560 x 50

= 28000 µg/ml

5. For the separation of these 4 proteins following column chromatography can be used:

a.)Affinity and gel filtration: Firstly the solution will be passed through the column of concanavalin A so that Protein C can bind with it. All other proteins will elute out from column. Then the remaining sample will be passed through gel filtration. So that proteins can be separated on the basis of their size. In gel filtration the larger protein will be eluted first as it will use void volume (space between molecules) to travel. So Protein A will be elute out first followed by D and B will be elute out in the end.

b.)Ion exchange chromatography: As we know the pI of all the proteins to be separated so ion exchange chromatography can be used. Below the pI, Proteins have overall positive charge and above the pI they contains overall negative charge. So firstly the pH of sample will kept above 7.8 so that protein A and D posses positive charge. On passing this sample to cation exchanger, protein B and C will elute out first and after changing the pH (decreasing upto 6.2) of eluent protein A and D will lose its charge and will be elute out from the column.

6.a. Proteins are amphoteric molecule but they have overall negative and positive charges. There is a pH of protein at which its overall charge becomes neutral and the protein gets precipitated. This pH is known as isoelectric point (pI). Below the pI the protein contains overall positive charge and above the pI it contains overall negative charge. So with the help of pI the charge on protein can be find out and they can be separated on the basis of charge on it.

6.b. In ion exchange chromatography the addition of acid or base is used to get the protein out of the column by neutralizing the charge over it so that the binding of protein from column can be break.

7. To determine the organelles of the cell that contains the protein density gradient centrifugation followed by western blotting can be used.

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