how to boil samples before load to SDS-PAGE? - (Jan/25/2008 )

I used to put my samples in boiling water for 5min.

And it used to work fine. But now, after boiling and centrifuge my cell lysate, I found a big pellet, which means a lot of proteins precipitated, right? and the volume of the sample shrinks too.

This time, I incubate the samples at 93 degree for 3 min. No pellet at all. I don't know 93 degree is good enough to denature proteins, will see....

so, what do you guys do to denature the protein samples?

Thanks!

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we used to boil the samples for 5 min., cool it and centrifuge it.

Now we just add sds buffer, sonicate it and centrifuge it.

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The main reasons for preparing the protein samples with SDS sample buffer before SDS-PAGE are to coat the proteins fairly evenly with the dodecyl moieties to allow a size dependent separation in SDS-page (as opposed to native PAGE where the charge of the native protein is a strong determinant), -and to reduce any sulfate bridges present.
Both are accomplished by heating. -and if you have a small sample volume 3minutes at 93 degree just might do the trick (as long as your lab is at low gegraphic altitude ;-)
Regarding you protein precipitating out: you wouldt happen to have KCl or phophate buffer in there? As both will precipitate with SDS the proper way to ensure a good separation in PAGE would be to first make a buffer change of your sample before adding the SDS buffer (precipitation or even better with a spinn column). -The easy way (unofficial and a bit like cheating) is to dilute you sample with water before adding the SDS sample buffer, that way you can somewhat awoid that the salts reach the conc nessecary for efficient complexformation.

Wishing you good luck and great data!
Dr.D.

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I agree with the precipitation thing here, with KCl and/or NaCl.. But the thing is that it seems to precipitate when he boils his samples.. which is pretty weird..

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when preparing total protein extract, i boiled samples 5' cool on ice 1' and spin before loading.
For nuclear extract, i got sometimes a pellet appearing in the sample. I noticed the pellet comes after cooling. so to get through this, i just put the tube 30'' on the heater before loading. The smaple temperature goes high again, but no evaporation occurs in this brief time. Amount is preserved and migration fine as checked by ponceau staining after protein transfert.

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instead of boiling, you can incubate at 65C for 10 minutes.

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Are you testing membrane protein? Bioling is not good for membrane protein because they get messed with lipids after biol and go into pellet. So for membrane protein test, we just add loading buffer and incubate in 37 degree for 5 mins.
Don't know whether this is related to your question, however that means warm incubation is enough for coating the sample with the SDS.

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Hi there,

I just left my proteins in the incubator (95 deg) for >30 min because of my dear allarm (*&%$!@^&$*#&#*%&!!!!!!). Do I have to discard everything?

Thanks for the help

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spin them down and see if your protein aggregated.

you can still try loading them.

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What's the difference between boiling the samples for 5 minutes and incubate them at 65ºC for 10 minutes? Which one would work better for low MW proteins?

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