InVision™ His-Tag In-Gel Staining Kit

This is likely due to overexposure - performing a longer exposure to detect low expression levels of the desired protein may result in staining of minor contaminants in the BenchMark His-tagged Protein Standard. Load less BenchMark His-tagged protein Standard or perform a short exposure to visualize and image the standard and then perform a longer exposure to visualize and image proteins expressed at low levels.

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

Here are possible causes and solutions:

- Missed washing steps. Be sure to wash the gel twice with 20 mM phosphate buffer. If the background is high, perform a third water wash step for 10 minutes.
- Poor water quality. Use ultrapure water (>18 megohm/cm) for washing and preparing phosphate buffer.
- Protein overloaded. Decrease the protein concentration or lower the sample volume.
- Dirty imaging platform. Always clean the imaging system with a paper towel prior to imaging the gel to minimize any background fluorescence.
- Non-specific bands. Highly basic proteins and divalent metal binding proteins such as carbonic anhydrase (30 kDa), SlyD (21 kDa), and phosphorylase B (97 kDa) may cross-react with the stain producing non-specific bands.

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

Here are possible causes and solutions:

- Inadequate staining. Use appropriate staining protocol based on the gel type. Use BenchMark His-tagged Protein Standard as a positive control to verify staining reagents and protocol. Avoid excessive washing of the gel.
- The gel is not visualized or imaged properly. Be sure to visualize the gel using a UV transilluminator equipped with a camera or a laser-based scanner using the correct filters (see manual for details). A Polaroid camera is not recommended. Make sure the aperture on the camera is open wide to allow enough light entry and that the camera is connected to imaging software that allows contrast adjustment for viewing the best image. Visualize the gel immediately after completing the washing steps. Storing the gel in phosphate buffer decreases the signal intensity.
- Low protein load or expression level. Check total protein content of the gel by staining the gel with a total protein stain (check page 13 of the manual). Load at least 1 pmole of the His-tagged fusion protein for detection. Make sure the His-tag is in-frame and the protein is expressed properly.

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

E-PAGE gels are thicker than standard mini-gels and result in too much background when stained with InVision His-Tag In-Gel Stain. To obtain better staining sensitivity, we recommend transferring proteins of E-PAGE gels onto a nitrocellulose membrane and then staining the blot with the InVision His-tag In-gel Stain as described on page 14 in the manual.

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

To perform western blotting after InVision His-Tag In-Gel staining of His-tagged fusion proteins:

- Record a permanent image of the gel after staining of His-tagged fusion proteins.
- Equilibrate the gel in 1X SDS Running Buffer for 1 hour.
- Perform western blotting and immunodetection using a method of choice.

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