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isoelctric gels hard to stain?


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#1 joy123

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Posted 22 January 2013 - 07:31 AM

Hi, I have been experiencing problems staining a commercial isoelectric focusing gel.

I followed the staining method recommended by company: fix in 12% trichloroacetic acid+3.5% sulfosalicylic acid for 30min, and stain with 0.1% coomassie R250 in 40% methonal and 10% acetic acid.

One paper used just 20% trichloroacetic acid to visualize the bands. Another paper used 0.1% coomassie R250 in 20% TCA (destain with 20% TCA).

It seems that my staining method was similar with theirs. I don't understand why I don't get the bands. Please let me know if you have suggestions. Thanks a lot!

#2 mdfenko

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Posted 23 January 2013 - 05:52 AM

how completely do you destain your gel (how is the background)?

ampholytes will interfere with staining so, if you are not using an immobilized pH gradient, you have to wash them out prior to staining (hence the tca-sulfosalisylic acid fixation step).

you may still have too little protein to detect with coomassie. have you tried silver staining?
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#3 joy123

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Posted 26 January 2013 - 08:10 AM

Thanks for your response!
The background was very faint after destaining.
Indeed, the articles used more sample than I did. I think maybe silver staining would be a good try.




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