Small peptides in gel - What to do to see a 2kDa peptide in coomassie gel (Aug/09/2006 )
You should first use Tris-Tricine buffer instead of Tris-Hcl and if you are working with precast gels Bio-Rad offers many alternatives.
Casting a gel for peptide separation is somewhat difficult because you should work with gradient gels.
I suggest you to work with pre-cast ready gels. You can visit the webpage of Bio-Rad
Hello people,What do you suggest to see a small 2kDa peptide in a gel, using Coomassie?
Please, help me.
Adri
Hi,
Do you have any further details about this? Do literally run everything as normal, but with double-strength running buffer? What about the buffers used to cast the gel... still the same I assume? Same current per gel?
Thanks.
I ran my 4kD peptide with 10%, 12% and 15% Tris-Tricine gel, but my peptide (from 10 ug to 40ug) could not show up in the gel with coomassie stain. My peptide contains 3 disulfide bonds and is very stable and very strong. It is active even after treatment with the extreme pH and boiling condition. I suspect that even after the treatment of reducing agent and boiling, my peptide still keep its secondary structure intact and can not run in the gel effeciently.
Does anyone have an idea how to treat this strong peptide before run it in gel? Thanks in advance!
http://depts.washington.edu/bakerpg/protoc...icine_gels.html
It works fine, however I modified the amount of ammonium persulfate in the stacking gel because it was shrinking. Instead of 100 µL I use 500 µL ( for 10 gels)
It's a 10% tris tricine, you might need to increase the percentage of acrylamide.
I don't remember exactly, but my lower band was around 3 kDa. Why don't you try 12 and 15%?
Does anyone have an idea how to treat this strong peptide before run it in gel? Thanks in advance!
http://depts.washington.edu/bakerpg/protoc...icine_gels.html
It works fine, however I modified the amount of ammonium persulfate in the stacking gel because it was shrinking. Instead of 100 µL I use 500 µL ( for 10 gels)
It's a 10% tris tricine, you might need to increase the percentage of acrylamide.
I don't remember exactly, but my lower band was around 3 kDa. Why don't you try 12 and 15%?
i assume there is also sds in the gel and sample buffer. then...
did you see anything at the top of the wells or at the stack-run interface?
how long did you boil? most don't boil for more than 5 minutes. you may have aggregated your peptide. you can try incubating the peptide, in sample buffer, at 65C for 10-20 minutes.
I treated my peptide with sample buffer which contains 4-8% SDS and 100mM DTT (too much?) and boiling for 5 minutes before I ran it in gel. After destaining, I only saw protein standard in the gel, other lanes were totally blank. Any suggestion? Do you think a urea gel will help? Thanks.
Does anyone have an idea how to treat this strong peptide before run it in gel? Thanks in advance!
i assume there is also sds in the gel and sample buffer. then...
did you see anything at the top of the wells or at the stack-run interface?
how long did you boil? most don't boil for more than 5 minutes. you may have aggregated your peptide. you can try incubating the peptide, in sample buffer, at 65C for 10-20 minutes.
the amount of sds and dtt seem to be okay (what is ratio of sample buffer to sample?). you may want to try heating at 65C for 10-20 minutes.
also try some other peptides and see how they run.
you could try adding some (6-8M) urea in your sample (with sds and dtt). in the gel, along with sds, urea may help sharpen bands.