Different levels of Protein over expression?

Differences in expression levels could be due to one or more reasons, but it's hard to nail down any particular one at this point, because your description is too vague. Your situation is not explained clearly--is it a single cell line for each, IOW, 1 separate cell line overexpressing each of the 3 different proteins, or each line overexpressing all 3 at the same time, or what??? Easier to give an intelligent answer if you provide this info.

EDIT#1: Ok, thanks, that helps, now here are some more questions to help narrow things down a bit more: 1) Are the different cell lines all from the same parent line, i.e. are they clonal, such that the only difference between them is the particular protein each is overexpressing? 2) Was each separate protein construct engineered into the same expression vector, or were different vectors used for each protein? 3) How large a difference in relative expression levels is there between each protein--ballpark amounts are sufficient, i.e. differences of 50% or less, 2-fold? 5-fold? 4) Is the DNA repair activity being examined in the cells themselves, or are you using extracts from the different lines in an in vitro repair system?

That's enough for now, your turn.

EDIT#2: You asked, "...the second bit confused me a little - the proteins were engineered into the same type of vector (not same individual vector) if that's what you're asking?" Yes, that's exactly what I meant. You said it better. Ok, that eliminates different cloning vectors for each protein, although it may still be possible that for one reason or another, efficiency of expression of a given vector may be affected by whatever insert is put into it--e.g. large inserts may express differently from smaller ones...another possibility is that the degradation rates may vary between the different expressed products, but beyond that I'm not certain.

When you do your assays, I assume you have tried to normalize the amount of material by running similar amounts of whole cell protein, right? But since your differences in expression are pretty large, that could cause you a problem, since in the 4-fold higher one, there's 4 times as much of the total protein that is active in the assay. At this point, the simplest thing you can do is use your data on relative expression levels, and either use similar amounts of ACTIVE PROTEIN (as opposed to just total protein) in your assay, or DO use similar amounts of total protein, but then correct the numbers for the amount of active product presumably present in each extract. If all 3 are about the same molecular weight, then you can use mass to normalize, but if they vary greatly in size, you'll want to compare them on a molar basis.

Another thing to consider might be trying to isolate and purify each one of your expressed proteins, which would be very easy to do if you can express them as a fusion to a His or GST tag, then add each one back in equimolar amounts to a control cell extract, that way you could more precisely control how much of each one is going into the assay.

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