why not boiled before electrophoresis? - (Nov/15/2006 )

Hi
I have some trouble with western blotting, the size of my protein is about 200KD, approximate 100ug nuclear extract were seprated in 6% gel, and then transferred for 3 hours at 50mA(5mA/cm2) , blocked with 5% nofat milk in TBST, added the primary antibody(diluted 1:100) overnight, washed three times, added the second antibody(diluted 1:500), detected with ECL kit.

but I had no band after x-ray exposure

and I noticed in a paper that they didn't boil the protein prior to electrophoresis , I dont know why not boiled before SDS-PAGE, if we do so, the electrophoresis rate is the same as boiled before electrophoresis? why we should boil the protein before electrophoresis?

thank you

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The aim of boiling is to help denaturation, but sometime some proteins aggregate
So, try not to boi and let's see

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thanks a lot

maybe I should try to incubate the protein with loading buffer in 60 degree for 30 minutes

If the protein are not denatured completely, the electrophoresis rate of the protein is different with which are denatured completely, isn't it?
If not boiled before electrophoresis , How can I confirm where is my protein in the gel?

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depends on the protein. Some make dimers...
How does it look on this paper?

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oh
It seems have similar electrophoresis rate with other papers showed. maybe I can try to do electrophoresis immediately after adding loading buffer, hope it can do.

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hi
i work with 150kD but see also the cellular form of 250kD of my protein
i boil 5' 95° and do the page.
do you check transfert by ponceau?
i don't detect my protein if the procedure is too short.

I work in semi dry transfert, so can't speak well with your references but :
2h at 15V --> nothing
overnight 5V (so very gentle) --> ok

so longer and slower may be your tip

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I transferred the protein for 12 hours, and I detected my protein. but the signal is very weak
I loaded 150ug nuclear extract. I don't know if the concentration of my protein is low.
the primary antibody 1:200, secondary antibody 1:1000

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thank you very much for that feed back
what are the cells you're currently using ? depending on cell types, promoter and the fact protein may be toxic or a disadvantage, you mauy recover less protein in a crde nuclear extract.

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My protein is a transcription factor. It has a high expression of mRNA in Hela, but the protein expression seems very low, both WB and immunochemstry shows very low protein expression. I don't know why.

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we work among others with HMw (>500 kDa) of muscle and never boil; instead incubate 1h at 30°C; as said, boiling accelerates denaturaration and opens binding sites for SDS but big proteins tend to aggregate

by the way, you confuse autoradiography with autoluminography

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