You could prepare samples from an uninduced culture as well as an induced culture. Any band on the Western blot that was common to both would be considered to be a contaminant.

You could prepare samples from an uninduced culture as well as an induced culture. Any band on the Western blot that was common to both would be considered to be a contaminant.

Hi there,

As anti his antibodies are raised against 6xHis containing peptides, signal from usual NiNTA contaminants will be marginal as they are indeed His rich but actually devoided of 6xHis sequence (but SlyD contains some consecutive His). Furthermore contaminants only copurify when the level of the protein of interest is low. MW is also helping to distinguish your favorite protein from contaminants except if they exhibit comparible MW. But as Adam mentioned, control culture should also help (I would also suggest empty plasmid transformed cells) in order to get the background.

I think before and after induction is a good approach, as well as loading different concentrations of the purified protein.

Hi,

I use this antibody

https://www.rndsystems.com/products/his-tag-antibody-ad1110r_mab050r#product_datasheets

to detect C-terminally His-tagged proteins expressed in BL21* strains. The advantage of this antibody is that I never observed any unspecific band until now. The disadvantage is the bad sensitivity, it is only applicable for strong overexpressed proteins, we identified proteins with a QExactive that were not detected with the antibody...nevertheless the signals observed were highly specific.

However I also always load a lysate from a strain transformed with the empty vector and same induction as the overexpression vectors.

Good luck