Tag Archives: experiment design

Gel bands and gel bandits

A protein ID confirmation is probably the most requested proteomics service in the facility; and it is not just a good (or ‘expensive’) idea – it could save you hours in the lab in the long run. When the MS result is not what you expected, don’t panic: use the MS information to your advantage! Knowing the parameters of interfering proteins (MW, pI, mechanism of metal ion binding) can help you to optimize or change your purification scheme.  

…Poly-His purification approach was inspired by high affinity of transition metal ions (divalent Co, Ni, Zn, and Cu) for His and Cys residues in naturally occurring proteins way back in 1975 …

What I usually see is a prominent gel band that is thought to contain a protein of interest with a poly-His purification tag which has been expressed in E.Coli and purified on an immobilized metal affinity chromatography (IMAC) column. When the protein of interest ID is confirmed, you leave me with a smile and a thank you, so read no further.

This post is for my disappointed customer whose gel band got hijacked by a bunch of E. Coli gel bandits. Having seen enough of their sneering mug shots/accession numbers, I have compiled a quick reference list of these interfering so-and-so’s from the references 1 and 2 (also included as pdf). Please note that these accession numbers are for K12, other strains will have a different accession number for the same gene product. Additional references, some of which offer solutions to the interference problem(s), are included for your enjoyment. If you encounter recurring interfering proteins in your purification system, and they are not listed here, please share this information!

Gel bandits reference list



  1. Bolanos-Garcia and Davies; Structural analysis and classification of native proteins from E. coli commonly co-purified by immobilised metal affinity chromatography. doi:10.1016/j.bbagen.2006.03.027
  2. Bartlow et al.; Identification of native Escherichia coli BL21 (DE3) proteins that bind to immobilized metal affinity chromatography under high imidazole conditions and use of 2D-DIGE to evaluate contamination pools with respect to recombinant protein expression level. doi:10.1016/j.pep.2011.04.021
  3. Robichon et al.;Engineering Escherichia coli BL21(DE3) derivative strains to minimize E.coli protein contamination after purification by immobilized metal affinity chromatography. doi:10.1128/AEM.00119-11
  4. Parsy et al.; Two-step method to isolate target recombinant protein from co-purified bacterial contaminant SlyD after immobilised metal affinity chromatography. doi:10.1016/j.jchromb.2007.03.046
  5. Block et al.; Immobilized-Metal Affinity Chromatography (IMAC): A Review. Methods in Enzymology, doi:10.1016/S0076-6879(09)63027-5

Gel bandits reference list pdf


Fingers crossed…

Photo by Evan-Amos

Photo by Evan-Amos

If someone told me “I honestly believe, the instrument is calibrated, it seemed okay last year when I checked it last time. It looks like the mobile phase is a bit old, I am pretty sure I replaced it 3 months ago when a spider fell into the bottle, so it should work. It is recommended to clean the ion source daily, but who has time, let’s hope it is not too dirty. I’m just going to run your samples and see what happens. Fingers crossed!” chances are, I would take my samples to be analyzed elsewhere.

So when I hear “We used old reagents, but they should still work, right? I found this old trypsin in our lab, I hope it is still okay. I am pretty sure the protein is still there, but it might have degraded. We bought new co-IP beads and are unsure how much protein we have loaded, but fingers crossed – everything worked!” I lose my mojo. If crossing fingers is a part of the experimental design, how are we going to troubleshoot when something goes wrong? Was it old trypsin or was the hypothesis no good?

Good news! There is a way to check if the old reagents still work and if the beads have the expected capacity – run a pilot experiment using something that always works; run a control experiment (or two) in parallel with the real experiment. Carry your control through the same procedure as your unknown. Check every step in the protocol: did it work? Our facility loves controls so much that there is no additional charge for analyzing them. So go ahead, bring as many controls as you think are necessary and you won’t have to cross your fingers!