Dear all
With respect to above mentioned subject, we use tricine SDS electrophoresis to resolve small peptides. Earlier separation was very good and getting sharp bands. Now we are facing problem that, bands are not good, we can see some smear bands. But standard protein and marker looks good.
We changed all the ingredients, we used chemicals of different lot numbers. Still we are getting smeared band for our sample. We can not use such gels to do western blotting also. Please anyone help to get rid of this.
Or suggest me any other method to analyse small peptides of 30 to 40 AA, qualitatively other tahn tricine sds page.
Thank you
if your marker and standard protein are looking good, it's probably not a problem of you gel but a problem of your samples. did you change anything in the sample processing protocol? Are the lysis buffers fresh,...?
if your marker and standard protein are looking good, it's probably not a problem of you gel but a problem of your samples. did you change anything in the sample processing protocol? Are the lysis buffers fresh,...?
Smearing can be a normal consequence of running membrane-associated proteins with a high lipid content in a discontinuous gel. In discontinuous PAGE, sample proteins are super-concentrated by a combination of two factors.
1. The rate of migration is suddenly slowed as the sample moves from a non-restrictive stacking gel to a separating gel that restricts movement.
2. (and more important), a pH change affects the electrophoretic mobility of proteins in the sample, causing the sample to check up abruptly. A sample of a half cm or so in height becomes compressed into a layer of bands a few micrometers thick, and the local concentration of protein goes up considerably.
Dear Ariane and Omar
Peptides are heterologous protein, expressed after cloning in shake flask. We used to harvest the media and analyse supernarent for peptide
Dear Ramanjini Gowda sir
We are loading very small amount of samples. And we are maintaining same volume of the samples from the beginning. Earlier we were getting good bands. But now bands are smeared.
Dear Rajalakshmi mam
Some amount of glycosylation is there. But earlier samples were more glycosylated than present samples, But still we could able to get good bands. Now the present samples are less glycosylated
You said that you are expressing after cloning in shake flask. If you are using animal cells do not shake them vigorously. shake very very gently, this may help by to saving breakage of cells
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