Trouble with Western Blot to detect secreted enzyme - what is causing the band pattern? (Apr/20/2008 )
Hi, I'm trying to detect the presence of an enzyme (endoglucanase, ~60kD) in supernatant (minimal media called Pliche), which my mentor believes is secreted by a particular fungus that infect plants, based on bioinformatic analysis. I am seeing a band pattern, and I don't understand what could cause it and how I can fix it.
The primary Ab was designed against a peptide with 10 amino acids, raised in rabbit, based on the bioinformatic analysis. It is thought this sequence would be at the active site of the endogluconase. I received serum.
There was nothing in the control or the ultra-filtrate; both of those are good. In the actual samples, there is a gradient of bands, so many bands that it is pretty much just a smooth gradient, strongest at the very top (heavy proteins), lightest at the bottom. The overall gradient is strongest in the undiluted lane, slightly less strong in the 1:10 lane, less strong in the 1:100 lane, and barely visible in the 1:1000 lane. There was practically no background noise.
I guess the serum is cross-reacting with all of those proteins. This is what I don't understand--the peptide was 10 residues long, so unless every one of those proteins has that peptide sequence, why should they appear as bands? How would the rabbit have so many other antibodies that just happen to react with almost every secreted protein of this totally unrelated organism?
Could the secreted protein somehow have gotten uniformly degraded, randomly hydrolyzed, to form polypeptides of varying molecular weights, each with varying amounts of the target 10 residue peptide? This could account for stronger signals for higher weights and lower signals for lower weights, if the higher weight proteins have more of the 10 residue sequence? I don't understand what is happening, and thus have no ideas how to fix it. Any help is greatly appreciated, sorry if this is a long post. If you want any more info about my protocol just ask.
If it helps, here is my protocol:
After concentrating the supernatant 24-fold using an Amicon centrifuge filter, I serially diluted the samples in ddh2o, then mixed those samples, plus control media, plus the flow-through ultrafiltrate from the Amicon filter (should not have enzyme), in a 1:1 proportion with Laemmli and 5% 2-mercaptoethanol, then boiled for 10 mins, and loaded in a 7.5% BioRad ReadyGel, along with a pre-stained marker. Transferred to a BioRad Immuno-PVDF, blocked overnight in PBS w/5% dry-nonfat milk. Washed 2x5 min with PBST. Shook for 1.5 hours with PBST and 5% milk with 1:100 of the rabbit serum. Washed 3x15 min with PBST. Shook for 1 hour with PBST and 5% milk with 1:1000 secondary anti-rabbit. Washed 3x15 min with PBST, then used ChemiGlow by AlphaInnotech for detection.
I would start with SDS-PAGE and classical Towbin-blotting; I hope you immunized more than 1 rabbit with your immunogenic peptide to select the best serum
by the way, try to present your question more condensed and to the point...
you are probably getting a lot of non-specific binding. i would try to do a series dilution of your primary antibody 1:100, 1:250, 1:500, 1;1000. A lot of the non-specific bands should go away then. It would also be helpful if you had something to use as a control so that you knew your antibody was reacting with antigen, such as a recombinant line that overexpressed the protein either in your species or in bacteria.