You purified protein X via affinity chromatography (no defiltration step perform
ID: 226470 • Letter: Y
Question
You purified protein X via affinity chromatography (no defiltration step performed) and ran an SDS-PAGE gel of the sample with a set of controls. Below is the result of your SDS-PAGE analysis. What is the benefit of a protein ladder/molecular weight marker in an SDS-PAGE gel? Describe what you can learn about the protein bands found in lanes 2, 3 and 4 based on the protein ladder bands. Positive and negative controls are absolutely essential to the validity of experimental data. In this gel above, which lanes contain the positive and negative controls? Describe the information you can deduce by comparing and contrasting the negative control and lane 2 protein bands. Describe the information you can deduce by comparing and contrasting the positive control lane and lane 2 protein bands. You don't know the molecular weight (MW) of protein X and you are not able to find that information in the scientific literature. The best way to determine the MW of a protein using an SDS-PAGE gel is to use the protein ladder bands to create a Log(MW) vs. Rf graph and calculate the MW from the line of best fit. What is the equation to calculate the Rf of a protein band? Make a table of the Log(MW) and the Rf values for all 5 protein ladder bands. Describe any trends you see in the table values. Sketch a scatter plot of the data (log of MW on the y-axis).Explanation / Answer
A. The protein ladder is used to determine the size of the unknown polypeptide. The ladder will show bands in various molecular weight range. Our protein ladder is showing a band at 170, 80, 58, 46 & 25 kda range. The lane 2 is showing 2 bands whose molecular weight can be determined by comparing it with the ladder.
Band 1 molecular weight is between 58 and 80 kda. It is equivalent to the molecular weight of purchased protein which can be determined from lane 3 (positive control).
Band 2 has a molecular weight equivalent to 46 kda which is the same as that of the elution buffer in lane 4 (negative control). Hence it can't be a protein.
From these interpretations it can be concluded that the purified protein is a quality one and it has the same molecular weight as that of the company product. There is no contamination in the protein sample.
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