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protein precipitate
Posted by: eduardelacroix (IP Hidden, New member, 2)
Date: February 7, 2006 06:03PM

Hi guys, I'm about to do an SDS-PAGE, but there's some insoluble precipitate in my samples which I can't get rid of.

I used the Roche RTS 100 kit to express the protein (~10kD, should be water-soluble). The expression mix looks clear. But when loading buffer is added and then heated at 95C for 10 mins, some precipitate is formed.

I tried to get rid of the precipitate by repeating the experiment and removing the salt with methanol:acetone before adding the loading buffer. However, there's still some precipitate. Do you think the pH of the buffer might perhaps be too close to the pI that the protein is not very soluble or is my protein not soluble anyway? Or is there some other reasons for it?

 

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Re: protein precipitate
Posted by: kum81 (IP Hidden, Junior member, 10)
Date: February 8, 2006 07:09AM

hi
have u tried to run the gel with the precipitate?
make a fresh set of sample buffer with appropriate pH ~6.8. boil at 95C for 5 min, cool and centrifuge at 10,000rpm for 10min and then load on the SDS PAGEand stain.


hope it helps

best wishes

 

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Re: protein precipitate
Posted by: shahid (IP Hidden, New member, 2)
Date: February 19, 2006 11:22PM

a number of proteins have this problem. if you could please with a heat treatment at slightly low temperature i.e 85C for 15 minutes and then check it out. I think it may not precipitate this way.

 

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Re: protein precipitate
Posted by: Scaffoldman (IP Hidden, New member, 2)
Date: February 20, 2006 01:48AM

I am also using the Roche RTS-100 to express 6 different proteins but so far (touch wood), did not encounter this problem. This may sound very simply but did you by any chance put the samples on ice after heating with sample buffer? SDS precipitates out in the cold. I made this mistake during my very first few protein gels and I have learned ever since! Just to rule out some very trivial possibilities

Scaffoldman

 

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