Backgrounds persistence and higher than target protein in western blot - Expressed in BL21(DE3) (Mar/21/2011 )
Can't remember off hand the exact procedure because my notes still in the lab...
I am using Ptorino Ni-TED 1000 column, using the method for native protein, but I sonicate without lysozyme.
Proceed with manufacturer's protocol.
Washing: wash buffer (W- with b-mercaptoethanol 30mM, then W-B with tween-20 0.2% (Thanks to mdfenko for the tip ), then W-B with 1mM Imidazole, then followed by just normal W-B. Finally is elution.
In the elution, you got 3 elutions. I use the second elutions for majority of my proteins. If WB detects nothing in my second elutions, I go back to the first elution which appears to be more dirty (first elution is still with some 25kb band from hell, no choice)
Since my second elution is not concentrate enough (I guess...) I concentrate ot and buffer exchange to 0.1M HEPES buffer it with vivaspin (took ages waiting for the spinning). I do a-repurification later with MagneHis beads, without modification. IT took nearly 24 hours continuous working in the lab...
The result: I got a single clean band in WB, but in silver stain, I still get 4 bands in background. Not sure what caused it.
If you are from Malaysia we can talk over the phone and do some further discussions, perhaps meet up and maybe a mini-collaboration to make the protein purification in final: 1 band in WB, 1 band in silver stain...
Well, thank you very much for all the details! i'm going to try to incorporate these changes to my system. I hope this finally solves the problem! Thanks again!
Do let us know the outcome
Hi, I`m back. I´ve tried doing washes with both b-mercaptoethanol and tween-20 (in separate washes)...and there was still the band from hell in coomasie stain (I didn't try WB, since I already saw it in the gel). I also saw the same band in the non-IPTG induced control. Granted, the band is quite faint, specially in comparison to the band of my protein, but I'm really trying for more purity (maybe that is the best I'll get...though I can't understand why I din't see it before). It does seem that there is some histidine rich-protein from e.coli I just can't get rid off. So...I'm sorry to be bothering again, but does anyone have any other ideas?. Anything will help. Thanks!
For me, I still got the phantom bands in my silver stain (although my protocols were different with yours) but it doesn't come out in WB.. so I think is fine for me...
Maybe you can try column purification such as FPLC, size exclusion etc...