Hi,
I have encountered a weird problem. I have been trying to express a protein fragment from bacteria. This fragment has an N-terminal HA and a C-terminal 6X His tag. Following expression, I purify the protein using Ni column. In coommassie I see nice band at the right size. When I blot with anti HA antibody, I see nice band. However, with anti-His antibodies, I do not! So far, I have tried three different anti-his antibodies and none of them could detect my protein. I always incorporate a positive control for the his blot (a separate protein that is also his tagged), and this control protein always shows up. I know that this is not an issue with regards to the following aspects:
1. Incorrect construct? - There is no mutation in the construct. Sequencing looks just as expected.
2. Something choppping off the protein in bacteria? - used protease inhibitor while lysis, same result.
3. In vitro translation? - Used the NEBpure express kit to in vtro translate the protein, same result as from bacteria expression.
4. Pull down with HA: Following in vitro translation pulled down the protein using anti-HA. Protein pull down worked fine, as nice band is seen while blotting for HA. However, again, bloting for His did not show any band.
Any suggestions on troubleshooting will be highly appreciated!
Thanks
Sumit
I'd try a spot-blot and see if the non-denatured protein reacts. Theoretically, if you can isolate the protein on a Ni-column, the His-tag should be present and accessible.
SDS-PAGE is about as denaturing as you can get (strong detergent and reducing agent). Just for the fun of it, you could try CTAB-PAGE (DOI:10.1016/S0003-2697(02)00639-5, perhaps even leave out the reducing agent), it sometimes works where SDS doesn't.
But as I said, I'd try if the non-denatured protein reacts, and take it from there.
I agree with both answers, when you purify your protein, Sure you do it under denatured conditions, making more accessible to bind with Ni2+. When you do yor Wstern blot, In the Blocking step, protein re-folding occurs and here is where your 6xHis tail hides. Alternatively you could try to degrade your protein and do the same western if you need so
There are several factors that may lead to variability of detection among different His-tagged recombinant proteins from dot blot to SDS PAGE( could be altered buy SDS revealing as no detection on wetern blot). These include the availability of the His-tag to the antibody (due to both primary and higher-order protein structure), the location of the His-tag on the individual protein, protein purity, antibody dissociation constant, and the length of the His-tag. The detection of the same His-tag on different proteins is not consistent generally, and is important for further characterization and analysis of His-tagged proteins and antibody selection. Furthermore, such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods
Please read the following paper:
Article Variability in the Immunodetection of His-tagged Recombinant Proteins
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