Protein Lab 5 - Void Volume & Blue Dextran

When discussing void volume in class, Bradshaw mentioned that most had a void volume around 12 ml.
Who had this? Group 1 fractions (2ml) were
1, 2, 3 clear
4,5,6,7, blue dextran which is above the exclusion limit

Is this correct? "Anything that is above the exclusion limit, comes out in the void volume. So we measure the void volume at the point where blue dextran has completely eluted."
I'm confused because I also have notes saying that "void volume is the volume that comes off CLEAR, prior to any molecules separated." (Argument: blue dextran is above the exclusion limit & is NOT part of the molecules (proteins would have been better worded?) being separated.)

I was confused on this also. She said the void volume was clear and came off before the blue dextran. I think we only used the blue dextran so that we could calculated the void volume and see the separation of the 2 different molecules.
I was wondering about question 6 A&B though. Would it still be that none of those molecules are in the void volume even though some are higher than the exclusion limit?

Keep it simple. For Question 6A and B the proteins that should come off with the void volume are those that are above the exclusion limit. The rest should come off in order of the highest molecular weight, assuming they are all globular.

The void volume in our experiment was the clear tubes of buffer that came off before the Blue Dextran. The part that is confusing is that the Blue Dextran itself is a big ass sugar that is usually above column exclusion limits, so in a technical sense it should come off with the "void volume". However, in our experiment the Blue Dextran is considered an analyte, and so it is not part of the void volume. The purpose was really just to show how the column works with two molecules, although I'm sure Blue Dextran was chosen because another protein would cost the lab more dinero

Hope this helps

That DID help. I think in the real world, blue dextran is used to determine the void volume: void volume would = the volume which contained the blue dextran. BUT, in lab, and as Bradshaw stated, the CLEAR fractions BEFORE any molecules separated = the void volume.

Sephadex info for notebooks:
Lot #
G50-150 61K1379 (Thanks Aly)
G100-120 ?? (Failed to collect)
G200-120 120G6204 ? (Is this what everybody has?)
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Sephadex for Report:
Water gain
G50-150 ??? N/A is this correct?
G100-120 10.0 +/- 1 gram
G200-120 20.0 +/- 2 gram

Thanks Kyle. That was how I was thinking it would be, but kept going back and forth as to whether it was right.
G100-120 lot # is 128C-0204.
Water gain for G50-150 was not given.

So, don't the proteins come off in the same order in both A & B, the only difference is which ones are considered part of the void volume?

Sllawson0 wrote:

So, don't the proteins come off in the same order in both A & B, the only difference is which ones are considered part of the void volume?

Correct. The only difference between the two resins is their exclusion limit, so there's an extra protein that won't interact with the column and should flow right through with the void volume.

What was the exclusion limit of the G200-120? I wrote above 100,000 in my lab notebook, but I think it should have been 200,000.

Sllawson0 wrote:

What was the exclusion limit of the G200-120? I wrote above 100,000 in my lab notebook, but I think it should have been 200,000.

It is > 200,000 in my notebook.

Gracias!!