I extracted whole protein from different cells and tissue and detected their concentration with Bradford assay (in several repeats they are highly concentrate, about 20-40 micrograms per microlitre). But when run it on sds-page gel, protein bands are very very faint with high background. In fact, bands are condense but don't stained. I changed my coomassie stain and refresh it but my problem is not solved.
Thank you for your answer but i know that my protein is very concentrate and before with this procedure and with loading about 20 micrograms per microlitre i get strong bands
if u r trying to stain ur gel wit colloidal CBB . we have two protocols where in we use aluminium sulphate in one and ammonium sulphate in the other ..try checkin the ammonium sulphate procedure. the intensify the faint bands.. coz u say ur protein is already concentrated this shud wrk out .
Dear Coleague,
In reference to suggestions of Professor Neuhoff, Göttingen long years ago, may I drive your attention to my paper published in J Clin Chem Clin Biochem 23 (11) 1985, 777-780
The best reult concerning sensitivity can be obtained when using Coomassie Blue R-250 (CBR) in Perchloric Acid. And, in almost sarurated concentration in as long-time as possible staining procedure. For this, you have to care about following points:
- the use of methanol in staining solution makes the method faster, but it produces more or less irreversible background darkening
- the sensitivity of staining increases considerabely with the staning time (ideal: over night)
- the best result is acheived when using at least 0,4g/L (or even over-satrurated) CBR in 35g/L perchloric acid while gentle shaking. Therafter you make destaining by diluted acetic acid, and, at the biginning of the first destaining step you gently brush out fine dust of the CBR from the surface of the gel...
Good Luck!
AL
i have some suggestion about SDS-gel running,
1-after collecting pellet fraction with lysis buffer,be sure use as same as volume of your bacteria pellet.it can effect on your band concentration.
2-run stacking gel in 75voltage,and come it upper until100V each1hours,and do it for resolving gel so,from 100v until140-
3-stain gel in coomasie blue solution on shacker(70rpm) for 1 hours,and destain it in methanol destaining for overnight.(make your destain soloution fereshly).after that you can destain gel with ethanole destaining for 1hours also.
good luck
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