InVision™ His-Tag In-Gel Stain - FAQs

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.

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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.

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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.

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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.

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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.

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Proteins stained with InVision His-Tag In-Gel Stain are compatible with mass spectrometry (MS) analysis.

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The manual has a protocol for staining His-tagged fusion proteins transferred onto a nitrocellulose membrane. This procedure is not recommended for staining PVDF membranes.

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The BenchMark His-tagged Protein Standard is ideal for use as a positive control for the InVision His-tag In-gel Stain. The standard is formulated to allow simultaneous detection of standard proteins and His-tagged fusion proteins. The BenchMark His-tagged Protein Standard is included in the InVision His-tag In-Gel Staining Kit (Cat. No. LC6033).

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To visualize His-tagged fusion protein bands after staining, you will need one of the following:

- UV transilluminator (302 nm) equipped with a camera capable of integration
- To view and photograph a gel on the UV transilluminator, use a standard video camera, CCD (charged couple device) camera, or a cooled CCD camera with ethidium bromide filter or band pass filter encompassing the emission maxima (590 nm) of the stain. A Polaroid camera is not recommended. Note: You can use 365 nm UV transilluminator, but you may have to expose the gel for a longer time, as the sensitivity is lower than using 302 nm UV transillumination.
- Laser-based scanner with a laser line that falls within the excitation maxima of the stain (560 nm), and a 560 nm long pass filter or a band pass filter centered near the emission maxima of 590 nm. The sensitivity of detection is 2-fold more with laser-based scanners than with UV transillumination.

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The maximum excitation wavelength for InVision His-tag In-gel Stain is at 560 nm and maximum emission wavelength is at 590 nm.

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The stain is capable of detecting approximately 0.5 pmole of a 6X His-tagged fusion protein (e.g., 1 pmole of a 30 kDa protein is 30 ng). The staining intensity is sensitive to the number of moles of protein contained in a protein band as 1 molecule of InVision His-tag In-Gel Stain binds to only 1 oligohistidine tag molecule of the protein. For example, if you load 150 ng/band of 2 proteins with a molecular weight of 150 kDa and 30 kDa, respectively, after staining with InVision His-tag In-Gel Stain, the 30 kDa band stains more intensely than the 150 kDa band. This is because there is only 1 pmole of the 150 kDa band while there are 5 pmoles of the 30 kDa band in the total mass loaded (150 ng/band). The staining of a mini gel is complete in less than 3 hours.

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The InVision His-Tag In-Gel Stain is pink in color.

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InVision His-tag In-gel Stain is a ready-to-use, proprietary fluorescent stain that is specially formulated for fast, sensitive, and specific detection of His-tagged fusion proteins. It consists of a proprietary fluorescent dye conjugated to Ni2+: nitrilotriacetic acid (NTA) complex. The Ni2+ binds specifically to the oligohisitidine domain of the His-tagged fusion protein allowing specific detection of His-tagged fusion proteins from a mixture of endogenous proteins.

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We recommend storing the InVision His-Tag In-Gel Stain at room temperature where it is stable for 6 months.

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