I did disassemble/reassemble treatment of HBc VLPs. I collected the disassembled protein fractions using Ion exchange chromatography and reassembled overnight in buffer (NaCl, Hepes, DTT). I made TEM grids, it showed nice reassembled VLPs. Then I concentrated the reassembled VLPs by spin column and measured the concentration by BCA assay. I loaded 100 ug of reassembled VLPs into 5 ml sucrose gradient (SG) which should be present at the middle the gradient. I analysed the SG fractions by SDS-PAGE. After running SDS page, I stained the gel with Coomassie brilliant blue R-250. When I de-stained, there was no band in the gel except positive control. I also did Western blot analysis using anti-HBc antibody which also showed the positive control and no other bands.
How can I resolve the problem? Would appreciate if you please let me know your valuable opinion.
Thank you in advance for your responses.
Are you sure that you would be able to see the protein with just coomassie staining? Depending on the size of your gradient and the fraction size, you would not expect to see much in each individual lane of your gel. It could be that your proteins got stuck in the well and never entered the resolving gel, but more likely you don't see them because coomassie is not sensitive enough. Have you tried silver staining?
You did not mention how you collected the band of protein from the sucrose gradient. How do you know you collected the protein? Even if you didn't miss it, it would have been greatly diluted, e.g. 100 µg in 1 ml (0.1 mg/ml). How much volume of that fraction did you load on the gel? If you loaded 10 µl, that would be 1 µg, which is easily detected by Coomassie Blue staining.
I suggest that you fractionate the sucrose gradient and load samples of every fraction on SDS-PAGE to make sure you don't miss the fraction. An alternative would be to do a protein assay on each fraction, although sensitivity may be insufficient if the protein is too dilute.
Coomassie staining is sensitive enough to detect as little as 0.1 µg if it is done carefully. If you need greater sensitivity, you could use a fluorescent stain, or silver stain, as suggested by Michael Adams.
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