I am working on an extracellular bacterial protein. The exact molecular weight of the protein is unknown but it ranges from ~13 to ~185 kDa depending upon bacterial species. I was trying to observe the protein band in SDS-PAGE using 5% stacking and 12% resolving gel. My protein concentration was found to be 450 ug/ml in Bradford assay. I prepared my samples by mixing the protein samples with 6X Laemmli buffer (5:1) followed by heating at 95°C for 5mins. However, after running the gel, I observed that maximum protein residue remained in the wells and not passing through the gel. After development (coomassie staining and destaining), I got a band at around 8 kDa. The rest of the resolving gel remained blank. I have repeated the experiment several times but every time I got a similar result. I am not understanding what is going wrong.
Can anyone suggest any solution for this?
If your protein is recombinant you should know the molecular weight of your protein from the amino acid sequence. Copy paste the protein sequence here:
https://web.expasy.org/protparam/
If you are trying to purify this protein natively and not recombinantly, you won't get high yield or purity. Coomassie R-250 staining detects about 5 micrograms of protein in a gel. I usually load upwards of 20 micrograms of protein into the gel. You should be loading 11 uL of your protein into the gel to see something.
If you don't see anything then you simply don't have enough protein for further use. If you just want to be sure if you have protein at all, you can stain your gel by silver staining instead. The procedure is more complicated but this method detects nanograms worth of protein so your first try you will probably overload the gel.
Use different conc of stacking and resolving gel. If the sample is not running try to increase the porosity of gel. Use different permutations combination with protein conc n gel porosity. N check the voltage. Always run the gel half initially to avoid over run.
It is very unusual that homologues of a protein in different species have such a wide mass range. However, a gradient gel of, say, 5-15% with a 4% stack should be able to resolve this. If you do not have a gradient maker, simply include 10% glycerol in the heavy solution and mix aliquots of heavy and light solution for a step gradient with 1% steps. The glycerol makes layering the steps easier, but has no effect on resolution. I'd use methylene blue for polymerisation (doi:10.1002/elps.1150170112), so I can start the polymerisation once the gradient has been pipetted.
Also, do make sure that the blue colour in the Bradford test really comes from protein and not some contaminant. Simple test: spot 1 µL of sample on a NC or PVDF membrane, wash and stain with amido black or similar.
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