isoelctric gels hard to stain?
Posted 22 January 2013 - 07:31 AM
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!
Posted 23 January 2013 - 05:52 AM
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?
genius does what it must
i do what i get paid to do
Posted 26 January 2013 - 08:10 AM
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.