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Invitrogen™ InVision™ His-Tag In-Gel Stain

Product Code. 11569076
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For Research Use Only. All usage must comply with product instructions.
Product Code. 11569076 Supplier Invitrogen™ Supplier No. LC6030

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For Research Use Only. All usage must comply with product instructions.

Description

Includes

InVision™ His-tag In-gel Stain is supplied as a 1X ready-to-use staining reagent.

One ready-to-use solution that does not require mixing, dilution, or solution preparation

InVision™ His-tag In-gel Stain is a ready-to-use, fast, sensitive and highly specific stain for visualizing His-tagged fusion protein bands directly in a polyacrylamide gel following electrophoresis.

  • Stain proteins fused to oligohistidine sequence
  • Eliminate western blotting-detect His-tagged proteins directly in gel
  • Detect nanogram levels of protein bands with a UV transilluminator, equipped with a standard camera, or a laser-based scanner
  • Visualize results in under 3 hours

Protein Gel Electrophoresis, Protein Gel Staining and Imaging, Proteins, Expression, Isolation and Analysis

TRUSTED_SUSTAINABILITY

Specifications

Content And Storage InVision™ His-tag In-gel Stain is supplied as a 1X ready-to-use staining reagent. Store at room temperature. Guaranteed stable for 6 months when properly stored.
Detection Location In-Gel Detection
Detection Method Fluorescence
Quantity 500 mL
Target Molecule Proteins (His-Tagged)
Product Line InVision
Product Type His Tag Proteins In-Gel Stain

Frequently Asked Questions (FAQs)

I used the InVision His-Tag In-Gel Stain to stain my His-Tagged fusion protein and used the BenchMark His-tagged protein standard as a positive control. I am seeing some additional bands with the BenchMark His-tagged Protein Standard. What went wrong?

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.

I used the InVision His-Tag In-Gel Stain and am getting high, uneven background. Can you please offer some tips?

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.

I used the InVision His-Tag In-Gel Stain and am getting very weak staining. Can you please offer some tips?

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.

Can I stain E-PAGE gels with InVision His-Tag In-Gel Stain?

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.

I have stained my His-tagged fusion protein with InVision His-Tag In-Gel Stain. Can I perform western blotting of the stained protein?

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.

Can I use a protein that has been stained with InVision His-Tag In-Gel Stain for mass spectrometry (MS) analysis?

Proteins stained with InVision His-Tag In-Gel Stain are compatible with mass spectrometry (MS) analysis.
Can I use InVision His-Tag In-Gel Stain to stain His-tagged fusion proteins transferred to nitrocellulose or PVDF membranes?

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.

Can you suggest a positive control to use with the InVision His-Tag In-Gel Stain?

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).

How can I visualize my His-tagged fusion protein after staining with InVision His-Tag In-Gel stain?

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.

What are the excitation and emission maxima for the InVision His-Tag In-Gel Stain?

The maximum excitation wavelength for InVision His-tag In-gel Stain is at 560 nm and maximum emission wavelength is at 590 nm.
What is the sensitivity of the Invision His-Tag In-Gel Stain?

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.
What is the color of the InVision His-Tag In-Gel Stain?

The InVision His-Tag In-Gel Stain is pink in color.
What is the composition of the InVision His-Tag In-Gel Stain?

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.

How should I store the InVision His-Tag In-Gel Stain and what is its shelf life?

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

For Research Use Only. Not for use in diagnostic procedures.

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