BL4820 Biochemical Techniques Lab - Week 8
NATIVE MOLECULAR WEIGHT OF SIGMA GOT
ESTIMATION OF NATIVE MW BY FPLC GEL FILTRATION
The native molecular weight (MW) of Sigma GOT will be estimated by gel filtration
chromatography. The data to do this is provided here. You should treated this data as if
you collected it in the lab running the FPLC system during your lab this week. I carried
out the experiment for you and the data I collected are below.
CALIBRATION OF THE FPLC GEL FILTRATION COLUMN
To estimate the MW of GOT, the gel filtration column must be calibrated using proteins of
known MW. . The standard proteins used for calibration and their native MW are:
| Protein | Native Molecular Weight (MW) |
| Alcohol Dehydrogenase (ADH) | 150,000 |
| Bovine Serum Albumin (BSA) | 66,000 |
| Carbonic Anhydrase (CA) | 29,000 |
| Cytochrome c (Cyt c) | 12,400 |
Estimation of GOT Native MW
Make a plot of the log MW versus ratio of Ve/Vo (like that shown above). Vo (void volume of the column) is equal to the time it takes Blue Dextran to elute from the column since it has a MW of more than 1 million. Ve is the elution volume of the various standard proteins and GOT. Measure these values from the data on the chromatograms shown below. Volumes are given in minutes, which is equivalent to volume since the FPLC gel filtration column is pumped at a constant flow rate of 0.5 ml per min. The total volume of the column is 24 ml so things eluting at 45 to 50 min are very small. A typical calibration curve obtained with the Sigma MW-GF-200 kit used for this experiment is shown below.
Experimental Data for Your Calculations:
To estimate the MW of GOT, calculate its Ve from the data shown below and use your plot of the standard proteins to obtain the log of GOT's MW, then convert this log to MW. If you do not have Semi-Log paper, then take log of MW of standards and plot on linear graph paper (as shown above).
Chromatogram for Blue Dextran (use to find Vo):
Chromatogram for 2 of the standard proteins (use to find Ve for BSA and Cyt c):
Chromatogram for 2 other standards (use to find Ve for ADH and CA):
Chromatogram for GOT (use to find Ve for GOT):
This GOT was purchased from Sigma Chemical Co. A student sample of GOT eluted from the FPLC at the same position as the Sigma GOT except it gave a much lower absorbance.
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