I am having problems regarding to protein separation via SDS-PAGE. As a newbie in this field, I have tried to optimize the conditions for protein induction. My protein size is in 20-25KDa range and for that I chose 15% gel concentration. I can see visible bands of desired protein (most probably) but their alignment with marker isn't same. They are stacked above the marker line. Any suggestion and explanation regarding this will be appreciated. Thanks.
Proteins don't always run at exactly the expected position on gels relative to markers due to variations in their amino acid composition that affects their mobility. Additionally, post-translational modifications can have an effect on mobility, especially glycosylation.
To convince yourself that you are looking at the right bands, you should run extracts from both uninduced and induced cells. A Western blot would also be helpful, if you have an antibody.
Proteins don't always run at exactly the expected position on gels relative to markers due to variations in their amino acid composition that affects their mobility. Additionally, post-translational modifications can have an effect on mobility, especially glycosylation.
To convince yourself that you are looking at the right bands, you should run extracts from both uninduced and induced cells. A Western blot would also be helpful, if you have an antibody.
To expand on Adam B Shapiro 's answer, yes most likely if you are looking at a relatively pure isolate and those are the only bands showing up, there is most likely a PTM (or more) that is causing a higher MW band shift. Unsure if you are only running an SDS-PAGE followed by a coomassie stain (will visualize all proteins on the gel, with low abundance proteins being hard to visualize) or are transferring followed by antibody detection (more reliable). If the latter is the case, consider the following:
Is there a signal/cleavage sequence to your protein that perhaps isn't being removed? Are you accounting for any tag that may also be there? Is the reducing condition in your sample buffer correct/is the sample prepared well?
Thank you so much for giving suggestion in simplest way. Can you please share any reference or literature which could help me in understanding the principle underlying. Adam B Shapiro
Thank you Reuben Samson . Isolate is a mixture and its just for initial indication about protein presence.
Here is one reference on the subject of anomalous migration of membrane proteins in SDS-PAGE.
https://www.pnas.org/content/106/6/1760
Sometimes your protein can run differently. I recommend you to study if it is a target for PTM or some cleavage.
Mehwish Iftikhar
As I understand from your question that your desired protein stacks above marker line. It should not happen , please try to repeat the experiment again treating your desired protein and marker under the same condition.
Or may be you should look at the procedure that how you have purified the protein, that could be the case too.
All the best!
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