Pierce™ 6xHis Protein Tag Stain Reagent Set - FAQs

This is likely due to weak cross-reaction staining of proteins containing histidine clusters. Here are our recommendations:

- Wash gel for additional time in water (step 5.)
- Slightly decrease staining time (step 2.)
- Adjust exposure time and other settings to minimize weak, non-specific staining.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are possible causes and solutions:

- Experimental protein not expressed at sufficient levels in the lysate being tested. Load more lysate per lane or otherwise check that the target protein is expressed at all.
- Experimental recombinant protein is not tagged with 6xHis. Check for presence of tag by an independent method (e.g., detection or purification by nickel-chelate chemistry.
- 6xHis tag on experimental protein is blocked by interfering substances in sample. Verify that nickel and other 6xHis-binding reagents were not brought forward from a previous step and use only high-quality water.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

Here are possible causes and solutions:

- Poor quality or insufficient exposure to appropriate UV-light source. If possible, use a CCD camera for detection; ensure that UV lamp delivers the appropriate wavelength for excitation (280-310 nm).
- Experimental protein is poorly expressed (insufficient loading). Insufficient protein was electrophoresed per lane for the detection method used.
- Insufficient washing; residual SDS in gel prevents binding of stain. Wash gel for 3 × 20 minutes in ultrapure water and restain .
- Experimental protein is small (less than 20kDa) and diffused from gel during washing step. Fix the gel 50% methanol:7% acetic acid for 15 minutes before performing the water wash.
- Poor diffusion of stain into gel. Increase staining time to 10 minutes (step 2); this may be repeated on the same gel.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The fluorescent signal is stable for several hours in gels stored in water. Signal may be detectable, if somewhat attenuated, after overnight storage.

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There is no fixation step in the Thermo Scientific 6xHis Protein Tag Stain Reagent Set staining procedure. Hence, staining with this kit does not inhibit subsequent total protein staining with general protein stains, or electrophoretic transfer to membrane. Note: Bis-Tris gels run in MOPS or MES buffer may require fixing in 50% methanol: 7% acetic acid for 15 minutes before performing the stain procedure. After electrophoresis, fix the gel and then proceed with Step 1 of the procedure.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

The Thermo Scientific 6xHis Protein Tag Stain Reagent Set can detect as low as 0.2 µg of a 35 kDa (5.7 pmol) His-tagged protein per band using a CCD camera, and as low as 2 µg (57 pmol) of the His-tagged protein per band with a UV transilluminator. Detection requires illumination of the stained gel with UV-light at a wavelength in the range 280-310 nm.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.

We recommend storing the Thermo Scientific 6xHis Protein Tag Stain Reagent Set at room temperature where it is stable for a year.

Find additional tips, troubleshooting help, and resources within our Protein Assays and Analysis Support Center.