I have a protein to be expressed with an apparent MW of 18.4 Kda.For that I used 13% resolving gel.I use an unstained protein ladder.After the run was over, I stained the gel with commasie.The bands above 50 Kda got stained well, both in the ladder and the induced and uninduced samples but the ladder and samples below it did not stain.What could e the reason?
Hi there,
Coomassie staining is efficient on any size of polypeptide provided the sequences contains the side chains interacting with the dye. Therefore the cut off size you are observing has to be due to the electrophoresis step itself. Are you spotting the migration front properly? I think a picture should be helpful for interpretation.
Shreyasi, i think you could not see the bands below 20 KDa it may be the problem of preparing the sample for loading . i also faced the same problem before. then i again prepared the sample with fresh loading dye and boiled well, then i have loaded then i could see the low molecular weight bands very clearly. try once
or it may be the gel not mixed well before casting. use fresh TGS buffer for ruuning.
good luck
Hi there...
I guess, you need to keep the gel in staining solution for longer period and destain for short period. That should work. The understaining problem may arise from many factors, the protein concentration is one of the factors followed by dye quality, gel quality, and incubation time.
I think if the protein marker contains the low molecular weight bands are separated properly but your protein is not separated properly means the problem with sample loading buffer and pH. please check that try again definitely you will get the band
Hi
I u need to standardized with protein conctn and volume with dye and need to know approx mol wt of ur protein. U have to prepare the gel conctn a/c to mol wt also then if mol wt of protein is of 20 KD to 80 KD --people go for 1 to 1 1/2 hr staining with coomassive and de staining with 5% glacial acetic acid for 1/2 hr.
- Badges
- Science method