I dis 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. Then I loaded 100 ug of reassembled VLPs into 5ml sucrose gradient which should be present at the middle the gradient. I analysed the 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?
Thank you in advance for your responses.
Hi Kaniz,
I would recommend you to try silver staining instead of CBBR250. Silver staining has higher sensitivity and you should be able to see your bands prominently.
If no band is present in your western blot, should not be able to get the band by the stain.
Could you run a western blot for each step sample to see whether you could detect it and to determine where you lose it?
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