1: Why do you think the column needed to be “dry” (there needed to be an the abs

Question

1: Why do you think the column needed to be “dry” (there needed to be an the absence of the buffer on the top of the Column bed) when the protein mix was loaded?

2: In your gel filtration experiment, which molecule, dextran blue or riboflavin, exited the Column first? Would thismolecule be the larger or smaller of the two?

3: Between the two experiments, when you run the mixture of dextran blue, hemoglobin, and riboflavin, on G75 and G25 column, which two compounds did not get separated? Why?

4: Native b -galactosidase elutes at 20ml. what elution volume do you expect for denatured b -galactosidase under identical experimental conditions.

Explanation / Answer

1. So that the protein would not mix with anything else other than the column bed so that it would not diffuse or dissolve in any other substance.

2.

The tube that contained the most hemoglobin and vitamin B12 total was our tube two because it was getting five drops of almost straight up protein mix without dilution of any sort and the least was the last one because there was barely any protein at all left.

3.

We needed to make sure that all the protein was out and that none would be stuck inside the column. It is almost as if we flushed it out.

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