You purified protein X via affinity chromatography (no diafiltration step perfor

Question

You purified protein X via affinity chromatography (no diafiltration 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.

Figure 1. SDS-PAGE of purified protein X. Lane 1, Protein ladder (in Daltons). Lane 2, purified protein X (affinity chromatography). Lane 3, purified protein X (company manufactured). Lane 4, elution buffer.

a. 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.

b. 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.

c. Describe the information you can deduce by comparing and contrasting the positive control lane and lane 2 protein bands.

d. 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. Protein markers and ladders provide a series of graduated mass standards in a single mixture. By running alongside a sample, protein ladders provide a reference to determine masses of all proteins within the analysed sample.

Protein bands found in lanes 2 tells that this sample contains two peptides, one is almost 46 daltons in mass, and the other is around 70 daltons in mass.

The band found in lane 3 tells that the sample contains only single peptide with approximate weight of 70 daltons.

Protein band found in lane 4 tells that the sample contains only single peptide with 46 daltons.

b. Sample applied in lane 1 is positive control, while samples applied in lanes 3 and 4 are negative controls.

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