Hi All,
I am trying to run gel for peptide dimers with molecular weight of 3 kDa. I want to see the peptides under reducing and non reducing condition. I have used a 16 % SDS gel and used both reducing and non reduced sample. I was able to see very low intensity band for the dimers even though I have loaded 20 ug of the sample. Is it because the peptide is acidic and the coomassie dye do not effectively stain the acidic peptides? if so, is there any way to stain acidic peptides in gel? How to run peptide of size lower than 3Kda as the dimers after reduction will run at around 1.5 KDa? Does Tricine gel help to solve this? Any help would be highly appreciated.
Thanks
Yes Tricine gel is for small proteins and peptides - you can use silver stain to detect minor bands
The Tris-tricine gel system is best for small peptides if you must run SDS-PAGE, but size exclusion chromatography on HPLC would probably be better. The peptides can then be detected by absorbance at 214 nm.
Tricine helps. Maybe even a gradient gel. And Silver staining ist MUCH more sensitive than Coomassie.
Hi,
Like Andreas wrote: try Tricine buffer and Ticine-gradient pre-cast gel from Bio-Rad (10-20%, e.g.: http://www.bio-rad.com/en-si/product/mini-protean-precast-gels/mini-protean-tris-tricine-precast-gels?pcp_loc=lnav) or Novex..
Wish u luck!
Thank you all for the suggestion regarding the gel to be used. It seems tricine gel fits well for my experiment. And HPLC for detection.
For identification you can cut band from gel and just do sequence it directly by Edman degradation, or do mass spectroscopy
I have tried to do 12% sds page for a lyophilized semi purified peptide samples stored for more than 10 months at -80c but i faced two issues:
1. no bands are seen or observed after staining the gel with coomassie dye i repeated the experiment many times no bands observed. plz can anyone suggest me what could be the reason for this problem.
2. when i add the coomassie dye to the sample it immediately loose its blue color and disappear and the sample will remain to have its original color. plz suggest me to over come this problem.
these peptides are isolated from plants. i even performed Bradford method for protein estimation and gives good result.
What was the composition of the sample when you lyophilized it? Anything non-volatile in the sample is still in it. These substances could be interfering with the Bradford assay, making it seem like there is more protein than there really is.
thank you Dr. Adam, but i removed tannins and chlorophyll, lipids, terpenoids, phenyl propanoids...etc. but i believe that still something is there. do you think that polysaccharides will interfere with the peptides. shall i run again sds page with silver staining. and if after this still i got nothing what your suggestions.
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