protein bands are not visible

[shumaila]

hi all.
currently i am working on different plant protein fractions already altrafiltered. but when i load say 25 ul of my sample with 5ul sample diluting buffer which contains mercaptoethanol, SDS, Tris-HCl and glycerol, bands are not visible in coomassie stain. i want to share with you people is there concentration issue that it is diluted?

looking forward to have kind suggessions.

[mmorgan]

With the coomassie stain protocol that I use it's difficult to see protein bands with less than 50ng of protein.

I always load Bovine Serum Albumin standards onto my coomassie gels to estimate the quantity of protein in any bands I see. If you run BSA controls and they don't stain, then something in the staining protocol is not working.

[samratlogan]

you maybe can try by protein precipitation by ethanol and what's the percentage of gel u did?

[shumaila]

i am also using precipitation in some of the samples but then another problem encounters me that the sample diluting buffer containing B-mercatoethanol is not enough to dissolve the ppts. incresaing the buffer volume, heating the sample every thing is done but even then some undissolve protein creates disturbance while loading and results in convective flows and vertical streaking. hence more heat is generated. i am using 10% resolving gel. can you share your recipe of sample buffer? may be i should improve that.

[shumaila]

staining protocol is working morgan but sometimes bands are faint due to less concentration, when i increased concentration the bands are streaked... i am using molecular wieght markers that are prestained.

[JennYeap]

Coomassie R250 is slightly more sensitive than G250, but the lowest concentration detectable is >15ng based on my experience. Alternatively, you can try silver staining, which is much more sensitive than coomassie staining; can detect as low as 1ng protein samples. for streaking issue, you can do a TCA/Acetone precipitation to purify your proteins before separation, this can also concentrate your proteins.

[skumarmadhu]

The endogenous proteases during ur extraction may be degrading protiens in ur extract
u try from the begining using this method
extract protien from plant material using 0.1M tris, pH 8.0 containing protease inhibitors n estimate using braford method, then load. load mininum 50 microgram protien for CCB stain.