SDS-PAGE running strangely - (Jun/17/2004 )

Hi
On SDS PAGE, the protein migrate depending on its MW not the charge because the SDS mask the charge. try to boil the protien in sample buffer, when I say boiling means, boiling at 100C and for at least 5 or 10 mins.

good luck

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I boiled it like you say, but it still migrate strangely like that and also in some published paper they reported about this.

Some proteins also migrate unexpectedly like that, but there must be some reasons for this.
I think what I need is the characteristics of the proteins migrate strangely so that I can know more about the structure and characteristic of my protein...
Could you please suggest anything else?

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Hi
ok. is this protein postranscriptionally modified, i.e, phosphorylated, glycosylated, this could change its migration pattern.
whats the gel composition, is it 10% or 12% acrylamide. current you run the gel at, or volts, all those are factors that could add to the confusion.
if you say that this is published that it runs at 37kD then whats the reason they say in this paper.

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Thank you, Mabusheh!
It is a mamalian protein, phosphorylated and myristoylated but I express it in Ecoli system, so it can not get post transcriptional modification.
The gel is 15%, 100 volts for 20 min and 200 volts for 40 mins.
The paper didnt suggest anything about this, just report like that.
Please help me figure the reason out...

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Hi
try 10% or 12% acrylamide gels, and try to run at 80 volts for 30 min, then at 50 or 60 volts for whatever time it takes for the tracking dye to run to the bottom of your gel.
I think expressing the mammalian protein in E.Coli changes the protien somehow, isn't that right? I think the protein synthesis machinary in bacteria adds up formyl methionine to the N terminus of the protein rather than methionine? I am not sure if there are other factors that could contribute to this. have you ever tried extracting this protein from mammalian tissue and run it on the gel and compare the differences.

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There is a paper talking about what you have mentioned:

FEBS Lett. 1984 May 7;170(1):81-4.
SDS-PAGE strongly overestimates the molecular masses of the neurofilament proteins.

I also have situation similar to you. My protein is about 12kDa, but it appears as about 16kDa in SDS PAGE. My protein has a flexible and very negatively charged C-terminal tail, so i think the tail may hinder the mobility. The negative charge residues may also affect SDS binding to protein, thus lower the mobility.

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My experience is that, as somebody already pointed out, the standard must be boiled before being used. What I do is to take 40 microliters from the stock, put this volume in a PCR tube, run a program - 100ªC for 5 minutes - and them let the solution reache the room temperature. It works fine. Another detail, my protein standards that AREN'T prestained always run better than the stained ones. Good luck.

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Problem solved already? I've had quite similar problems (not being able to get my protein of interest to where it should be) with my SDS-PAGE. I've tried a lot of different conditions and ways to pretreat my sample, none successful so far.

Have you tried running your protein in 8M UREA gel? With smaller proteins that could give extra resolution. Another thing could be the marker , have tried using two different markers (different manufacturers) on same gel (also stained and prestained). I wouldn't use too much time on running SDS-PAGE, eventhough it's a good and basic method, you still can't accuratedly determine protein size with it.

How do you know that you have really the right stuff there, can it be some other protein? Is sequencing possible (Edman or mass spec.)? Do you have a tag in your protein? I've been told that His-tag would alter the protein mobility.

Even though it's told in the books that the ratio of SDS/protein is 1.4g/g, I wouldn't take it too literally. Proteins still react differently towards SDS, there are known SDS stabile proteins or protein complexes. Phosphates for example in proteins are known to alter the mobility of the protein (and I mean more than ~80 Da). I would still run couple of gels, but I wouldn't sacrifice all the sample for this method. If you have an antibody against the protein, check that it recognizes your protein. If so then try mass spec. and peptide fingerprinting to see if there is some modifications or extra. I wouldn't be surprised if there isn't anything extra there.

Good luck with your study!

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Have you checked that your marker is running properly. I imagine this can be one trouble shoot that if your marker is degraded than it will run little below and showing that your protein of interest is at higher molecular weitht. (I am writing it as i faced something of this sort, markers can really put you in trouble.)

Run a positive control for the protein.

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similar question to you all, i have been doing a western on breast cancer cell lines looking for a specific gene expected to run at around 24 kD- i have found the expression level within the cell lines to be as i predicted, however, the bands show up around 60kD. i used premade gels 4-12%, new buffs, new ab, and have denatured the proteins at varying temps from 65-95 degrees... is there an explanation for this apparent higher protein size?

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