Our laboratory focuses on genetic and proteomic analysis of parasite-host cell interactions. As such, we rely heavily on the expression of recombinant proteins for the development of antigen-specific antibodies for immunoassays as well as to test for protein-protein interactions by affinity blotting. Recombinant protein expression involves the use of various plasmid expression vectors that often incorporate either an N- or C-terminal fusion protein containing a 6xHis epitope (or other tags). 6xHis epitopes facilitate the quick retrieval of recombinant proteins in solution through the use of Nickel-columns or “His-grab” plates (for higher throughput). During the process of optimizing the protocol for expression of a protein of interest, it is necessary to determine that the 6xHis epitope is indeed present on the expressed protein (i.e. confirm that the recombinant protein was not truncated at the C-terminus resulting in a highly expressed protein without a 6xHis tag). Classical methods require transferring the proteins from a gel to a membrane and probing with tag-specific (i.e. anti-His) antibodies. In an effort to reduce the amount of time involved in simply confirming the presence of a His tag on recombinant proteins, Pierce developed the GelCode 6xHis [In-gel] Protein Tag Staining Kit. The stain allows for UV detection (on a standard 280-330 nm transilluminator) of His-tagged proteins in a gel. Clearly, this protocol would allow for rapid screening of recombinant proteins that were expressed under varying conditions of time, temperature, inducer concentration, etc.
Use of the reagent is simple and straightforward. After gel electrophoresis, the gel is washed two times for 15-20 min in Milli-Q water. The blot is then incubated for a minimum of 5-10 minutes with the His Tag stain (with agitation). After two more 15 min washes with Milli-Q, the stain developer is added for 15-20 mins. After two more washes, the gel is ready to be viewed under UV. The appearance of faint, yellow bands identifies those proteins that contain a 6xHis tag. The gel can then be stained with Coomassie blue to visualize individual protein bands without the need for additional washes/steps. However, despite the ease of use and theoretical sensitivity of this reagent, in our experience, we have yet to detect highly expressed fusion proteins containing both a C-terminus 6xHis epitope and an N-terminus His-patch (see Figure). The presence of the His tag was confirmed by immunoblotting and detection using classical methods. Note that even in our repeated, failed attempts much more protein was loaded into the gel than the claimed sensitivity stated in the manufacturer’s protocol (0.2 µg or 5.7 pmol of a 35 KDa protein). Moreover, we had used a CCD camera to visualize the bands in the gel (which is purportedly more sensitive than visualization simply on a UV box and captured on film). We have since discontinued the use of this reagent in the lab since none of the optimization steps (as suggested by the manufacturer) proved helpful.
Faculty Research Associate
Department of Molecular Microbiology & Immunology
Johns Hopkins Bloomberg School of Public Health