Western Blotting Problem - High band (Jul/10/2004 )
Hi. I am struggling with Western blotting for small proteoglycans. I am getting an unexpected band at around 100kd, then after waiting for a long period a less obvious band appears around 45kd (where I'd expect it). I am first enzyme treating the samples with chondroitinase ABC plus Keratanase/Keratanase II or chondABC alone, which should result in smaller sized bands for the former...however, this is not happening and in fact I'm getting slightly higher sized bands with the multiple treatments?? I am using 10% Tris Glycine gels @ 125V, reducing agent ME (2 -5 %), boiling water bath for 5 minutes, nitrocellulose membrane, blocking with 5%BSA. The precision plus standards seem to be running just fine. Is there some kind of aggregation problem I'm experiencing or possibly a re-oxidizing/incomplete reduction phenomenon?? I thought my antibody, enzyme, sample buffer or ME may 'off', but changing each has not helped. Any advice or suggestions appreciated. Allan.
Sorry for such a lame reply, but does the literature site dimerization as an issue? A proteoglycan I am working on is apparently an SDS and reductant resistant dimer. The 35 kd theoretical weight is never seen on a gel even when produced in bacteria, only a 70kd band.