I am trying to separate a 40 & 43 kda protein. I run 6M urea sds page for that. But most of the proteins get stuck around the interface of resoving and stacking gel. The small amount of protein that manages to migrate is not sufficient to be detected by WB. The pre stained marker migrates smoothly. Please help.
Hi Ayan, this problem disserves a lot of questions.
1. Do your proteins have free sulfhydryls?
2. Are they from inclusion bodies?
3. Do you boil your samples?
4. Have you tried boiling the samples with BME plus urea?
5. What percentage gel are you using to separate 40kDa and 43kDa proteins?
6. Are they different proteins or different products from the same protein?
Hi Steingrimur, thanks for you reply.
1.No,they do not seem to have free sulphydryls.
2.They are cytosolic proteins. I am lysing the cells to extract them.
3.Yes, I do boil my samples at 100 degree Cel for 10 mints.
4. I only add BME to my sample but not urea.
5. I am using a 10% gel.
6. They are different version of the same protein.
I am attaching an image of the gel (10% urea sds gel) run with the same samples and then coomasie stained.
Mark 1 is the stucking of the proteins at the interface.
Mark 2 is something that I can see even through naked eye when the gel is running as that area swells up on the gel.
Hi Ayan, Your gel looks fine. You will always have some proteins that barely enter the gel when you are running cell lysates.
So the only assay you have for this protein is WB and you are not seeing any reactivity in the ~40kDa range, but you are seeing reactivity by WB only at the interface?
If your antibody does not recognize bands visible by Coomassie, then I am afraid that your antibody sucks.
Yeah, I am trying to do WB mainly..but can you throw some light why tge gel swells up around 'mark 2'. I can practically see that without even staining!
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