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Position: Discussion forums > Bioscience biotechniques discussion forums > Molecular biology troubleshooting forum
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RE:no bands after developing
Posted by: Mette (IP Hidden, New member, 0)
Date: August 9, 2004 05:20AM
> >
> > Hi > > > > I have used a stained gel[with Commassie] for western > blot > > wet transfer.I am confident that bands transferred on to > the > > membrane since I can visibly see them as the gel was > > stained. > > > > However, after Ab & secondary Ab treatments with washings > & > > ECL kit ,I see absoultely nothing on my X-ray film. > > > > I have tried 15s,30s,1m,5m,15m exposures. > > I used HRPconjugated Ab's. > > Both primary & secondary Ab dilutions have been tried in > 5% > > non fat dried milk in TBS-T & also in 0.05% milk in > > TBS-T.Blocking agent is 5% non fat dried milk. > > > > Any inputs on this one???? > > > > Thank you > > Sari > > > > hi sari, > i have had the same problem. maybe your AB are not specific > enough. Also, see if your ECL kit is a little old. > Hi sari Probably it is not a good idea to stain the gel with coomassie prior to blotting. It could block the binding of your antibody. I always stain my blot with ponceau after transfer, which is a much more gentle stain for proteins and it is reversible. Another possibility is to stain your gel with coomassie and then cut out the band of interest. Put it in 50% ethanol for 2 days in the freezer (this will shrink the gelslice) and transfer the gelslice to another polyacrylamide gel. During the electrophoresis the protein will migrate out of the gelslice and into your gel. After electrophoresis is complete, you can blot your gel and perform the western as usual. This has worked for me using NBT/BCIP development. You can even perform an 'in gel digest' using an enzyme that cuts 1-4 times within your protein and get fragments that will still be recognized by your antibody. In this way, you have an even stronger indication that your antibody is actually binding to your protein of interest. Good luck Mette
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