I am conducting an experiment on proteins for turbid water coagulation. I purified protein using Ion exchange chromatography and want to check the purity on SDS page. After running SDS page, I stained the gel with comassie brilliant blue G and when I de-stained, bands did not appear except for the marker. How can I resolve the problem? Thank you in advance for your responses.
Hi Moa
Is it possible that your protein concentration is simply to low in the bands? Silver staining might help you out, since it is more sensitive.
Hi Moa
Is it possible that your protein concentration is simply to low in the bands? Silver staining might help you out, since it is more sensitive.
I'd be worried about the protein concentration too. Your protein quantification (e.g. Bradford, BCA, etc.) may be off for some reason (e.g. detergents for Bradford, 2-ME for BCA). I'd run out known quantities of BSA as a loading control before worrying about the bands not being visible. I assume your marker is pre-stained, and as such cannot be used as a good indicator of staining efficiency.
https://tools.lifetechnologies.com/content/sfs/brochures/TR0068-Protein-assay-compatibility.pdf
Perhaps the composition of your SDS gel, basically its percentage is too low or too high for the size of your protein? For instance, one of my colleagues is working on a huge protein which does not migrate into the stacking gel at all but sticks to the pocket instead. So does the size of your protein match the range of the protein ladder and is the protein ladder resolved appropriately?
May be you should try Comassie brilliant blue R-250 for staining. I use the same in my lab
These are all excellent responses. There are only a few options. One is that your protein concentration of protein is too low. One is that the protein is stuck in the well. One is that the protein ran off the gel. Normally, if your molecular weight markers run nicely, then you know where the proteins should show up on the gel. However, you mentioned prior ion exchange chromatography. Did you desalt the protein(s) before running the gel? That is extremely important. When you concentrate the ion exchange fractions you typically concentrate a lot of salt, too. The salt will prevent your samples from being exposed to the correct electric field strength, which could explain their staying in the wells (as could aggregation). You can use a sephadex column (correct MW cutoff and properly prepared) or spin columns or dialysis/microdialysis, whichever fits for the quantity of material and budget you have. Follow the manufacturer's instructions or technical bulletin on preparing all of the desalting columns for use or they won't work. Best of luck, and apologies if this was obvious to you.
Hi Moa
Verify the following to come out of your problem
1. Fraction from ion exchange column should be checked for salt removal by dialysis .
2. Probably protein might have come out of the gel
3. You have not mentioned about amount of protein applied for SDS -PAGE
As other people already mentioned, I'm afraid that your protein concentration is not enough to be detected on SDS gel. Have you measure the protein concentration before running the gel? You couls try to stain the gel by using silver stain (it can be done on the same gel you stained with Coomassie. Coomassie G-250 and R-250 can be used. The first one is more sensitive and the second one provides better resolution and lower cost
Failure for the band to show up in SDS gel are due to commonly low concentration of proten. In my experience, running SDS sample after ion exchange chromatography, without desalting will make the band smile ( band diffuse) esp fow the low mol wt bands.
Not knowing much about your protein and the exact protocol of staining (but I assume is it the classic CoomassieG stain/destain in acetic acid/(m)ethanol), I can advise you to check the pI of the protein. I have observed poor staining of very acidic proteins in the past. In some cases the proteins were visible as negative colour (i.e. transparent) during the staining phase.
Also, as others pointed out, should this be the case or should the concentration of the protein be very small, you can try silver staining or other dyes such as colloidal coomassie, sypro-orange etc...
Other things you can try are to enrich your sample by precipitation (with TCA for example) or concentrating the sample before loading by "boiling".
HTH,
Marco
I agree with most of the speculations mentioned to address your questions. You need to provide sufficient information to get relevant comments and answers to fix your problems. Many factors can contribute to your problem. But you did not provide sufficient information for the scientific community to address your problems.
Thank you all for your genuine responses! I found that the problem was due to desalting and low concentration of protein.
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