I performed SDS-PAGE gel assay and found that only four protein marker bands are visible instead of seven! I am confused about the size of marker bands which have disappeared. I have repeated the process and have got the same result. What could be the possible reason? protein marker degradation?
If by "SDS-PAGE gel assay" you mean a single, denaturing polyacrylamide gel separation of proteins then you're likely destaining for too long or your stain preparation is old or has gone off a little. Much like agarose gel electroporesis of nucleic acids - signal derived from proteins in stained SDS-PAGE separated proteins, including the marker bands, is a function of protein size. The bigger the protein the more stain can incorporate and yield signal. So in this situation I'm assuming the low molecular weight bands are the ones that you're losing.
You could try using a different stain, such as Blue-Silver stain. You can make this yourself and it has nearly no background after destaining. This makes seeing protein bands quite easy even for low abundance proteins.
But if by "SDS-PAGE gel assay" you mean a Western transfer - then I'm guessing you're losing the higher molecular weight bands in your marker. This is typical as it takes longer for those proteins to transfer to the blot when compared to smaller proteins. Simply extend the transfer period.
Best of luck.
Thank you all for your valuable suggestions.
@Hong Yu- I am staining the gel directly after PAGE. I might be losing the low molecular weight markers as suggested by Dylan. I am using 10ul aliquots to avoid degradation by frequent thaw and freeze process. I have tried to avoid all the possible mistakes that could lead to degradation by checking the buffers, staining solution.
Could it be the case that the low MW bands have ran out of the gel? What percent do you use, what voltage and for how long do you run it?
You can try one of the multiple colors markers (e.g. http://www.taq-dna.com/protein-marker-xxl-deluxe-_136.html)
And if indeed you mean Western blot, and you miss the markers after transfer, try placing two membranes rather then one and see if your markers had passed to the membrane further away from the gel, suggesting the transfer is too long. Also, stain your gel after transfer and see if all the marker has left the gel.
Yoram
I would recommend making the gel concentration gradient. It could be 7-20 and cut the run as soon as the front reaches the end
@Gaurav Kumar Singh , I believe it is a gel concentration problem , if your gel concentration is too high , high marker can't gos down throught the gel , and then if your gel concentration is too low , low marker gos over the gel and into the buffer. So try to adjust the gel concentration, get a good luck.
maybe there is something wrong with your SDS? Instead of SDS-PAGE you have simple PAGE. In that case the protein marker is not separated accroding to it's mass but to mass-to-conformation way. Then you may see only 4 instead of 7 bands (if it's ThermoScientific Prestained Page Ruler that exactly what happens). Are your bands sharp or a little smudged? If they are smudged change SDS solution :)
Thanx Marta. I had problem with SDS solution. I changed the solution. Now the bands are more sharp and distinct.
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