Five standard proteins were used to calibrate an SDS-PAGE molecular weight deter

ID: 180171 • Letter: F

Question

Five standard proteins were used to calibrate an SDS-PAGE molecular weight determination. The distance traveled for each of these proteins, the dye front, and the molecular weights are: A purified protein was examined, after reduction and alkylation, in the same SDS-PAGE system as used for the standard proteins. This protein gave two bands, of equal intensity, with R_f values of 0145 and 0.550. aOn an SDS-PAGE system, the molecular weight is related to R_f by the relationship: log(MolWt) = -AR_f + B where A and B are constants determined by the acrylamide concentration and the experimental setup. The native protein was chromatographed on a gel filtration column, with a void volume of 19.7mL, and a total volume of 91.2mL. A single peak of protein eluted with an elution volume of 32.6mL. On the same column a series of molecular weight standard proteins eluted at the following volumes: For the particular gel filtration column, Kav is related to the molecular weight by the equation: K_av = -Alog(MolWt) + B Where A and B are constants, and MolWt is the molecular weight. (K_av = {V_e - V_o}/{V_t - V_o}} a) Calculate the molecular weight of the native protein. b) Propose a compositional structure for the protein.

Explanation / Answer

We can also make graph to calculate molecular weight of desired Rf value

For this we have to find the logarithm value of molecular weight and take them on y axis. Put Rf value of x axis.

Molecular weight

logarithm value log10

97700

4.99

56000

4.75

35500

4.55

26000

4.41

14000

4.15

From the above plot we can find out the molecular weight of 0.145 and 0.550 Rf protein by calculating its anti log value.

For 0.145, the protein lies between 4.5 and 5.0 of Y axis so we can take 4.75 as our log MW value.

Antilog 4.75 = 104.75

56234 dalton

The protein contain atleast number of residues = 56234/ 110 511 which have maximum number of negative charged amino acids to make its pH around 4.7