InVision™ His-Tag In-Gel Staining Kit - FAQs

这可能是因为曝光过度——为检测低表达水平的目标蛋白而加长曝光时间，可能导致BenchMark His标签蛋白标准品中的微量污染物显色。可降低BenchMark His标签蛋白标准品的上样量，或者先以较短的曝光时间对标准品进行成像，然后以较长的曝光时间对低表达水平蛋白进行成像。

以下是可能原因和解决方案：

•遗漏了洗涤步骤。应使用20 mM磷酸盐缓冲液洗涤凝胶2次。如果背景很高，应进行第3次水洗并洗涤10分钟。 •水质较差。应使用超纯水（电阻率大于18 megohm/cm）洗涤和制备磷酸盐缓冲液。
•蛋白质上样量过多。降低蛋白浓度或减少上样体积。
•成像平台不干净。每次进行凝胶成像前都应使用纸巾清洁成像系统，以最大程度的降低背景荧光。
•非特异性条带。强碱性蛋白和二价金属结合蛋白，如碳酸酐酶（30 kDa）、SlyD（21 kDa）和磷酸化酶 B（97 kDa），可与染色剂发生交叉反应，产生非特异性条带。

以下是可能原因和解决方案：

•染色不充分。应使用适合所用凝胶类型的染色方案。使用BenchMark His标签蛋白质标准品作为阳性对照，对染色试剂和方案进行验证。避免过度洗涤凝胶。
•凝胶显色或成像不恰当。应使用带有照相机的紫外透照仪和具有正确滤镜的激光扫描仪（详情见使用手册）进行凝胶成像。不建议使用Polaroid照相机。应确保照相机光圈打开并有足够的光进入，并且照相机应连接到可调整对比度的成像软件上，从而获得最佳的图像。完成洗涤步骤后，应立即对凝胶成像。将凝胶保存在磷酸盐缓冲液中，会降低信号强度。
•蛋白上样量或表达水平较低。应使用总蛋白染色剂对凝胶进行染色，检测凝胶的总蛋白含量（见使用手册第13页）。至少上样1 pmole的His标签融合蛋白进行检测。应确保His标签是框内融合[读码框正确]，并且蛋白表达正常。

由于E-PAGE凝胶比标准小型凝胶更厚，因此，使用InVision His-Tag In-Gel Stain染色会产生过强的背景。为获得更好的染色灵敏度，我们建议先将E-PAGE凝胶上的蛋白质转印到硝化纤维素膜上，然后根据使用手册第14页的说明使用InVision His-tag In-gel Stain对印迹膜进行染色。

为了将经过InVision His-Tag In-Gel Stain染色的His标签融合蛋白用于免疫印迹分析，应该

•在His标签融合蛋白染色后，记录凝胶的永久性图像
•将凝胶置于1X SDS电泳缓冲液中平衡1小时。
•使用所选方法进行免疫印迹分析和免疫检测。

经过InVision His-Tag In-Gel Stain染色的蛋白质可以用于质谱（MS）分析。

使用手册中有对转印到硝化纤维素膜上的His标签融合蛋白进行染色的实验方案。不建议将该方案用于PVDF膜染色。

BenchMark His标签蛋白标准品非常适合作为InVision His-Tag In-Gel Stain的阳性对照。该标准品的配方能够同时检测标准蛋白和His标签融合蛋白。InVision His-tag In-Gel Staining 试剂盒（货号LC6033）中包含BenchMark His标签蛋白标准品。

为了在染色后观察His标签融合蛋白，您将需要：

•配有集成相机的紫外透照仪（302 nm）——为了在紫外透照仪上观察凝胶并拍照，可使用标准摄影机、CCD（电荷耦合器件）相机或冷却型CCD相机，并带有溴化乙锭滤光片或包含染料最大发射波长（590 nm）的带通滤波片。不建议使用Polaroid相机。注意：您也可使用365 nm紫外透照仪，但是需要延长凝胶的曝光时间，因为其灵敏度低于302nm紫外透照仪。

•激光扫描仪，带有属于染料最大激发波长（560 nm）的激光线，以及560 nm长通滤波片或以590nm最大发射波长为中心的带通滤波片。激光扫描仪的检测灵敏度比紫外透照仪强2倍。

InVision His-tag In-gel Stain的最大激发波长为560 nm，最大发射波长为590 nm。

该染料可检测到约为0.5 pmole的6X His标签融合蛋白（如1 pmole的30 kDa蛋白质为30 ng）。染色强度对蛋白质条带所含的蛋白质摩尔量很灵敏，因为1分子的InVision His-tag In-Gel Stain只能结合蛋白质上的1个寡聚组氨酸标签分子。例如，分子量分别为150 kDa和30 kDa的蛋白质上样量均为150 ng/条带，使用InVision His-tag In-Gel Stain染色后，30 kDa条带的染色强度高于150 kDa条带。这是因为，在150 ng/条带的相同上样总质量中，150 kDa条带只有1 pmole，而30 kDa条带有5 pmole。小型凝胶的染色在3小时内即可完成。

InVision His-tag In-gel Stain是一种即用型专用荧光染料，其配方专用于快速、灵敏和特异性检测His标签融合蛋白。该染料是由独特的荧光染料与Ni2+:次氮基三乙酸（NTA）复合物结合而成。Ni2+可特异性结合His标签融合蛋白的寡聚组氨酸结构域，从而能够特异性检测内源蛋白混合物外的His标签融合蛋白。

InVision His-Tag In-Gel Stain可在室温下稳定保存6个月。

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.

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

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

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.

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

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).

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

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.

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

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

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

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.

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

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

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

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.

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

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

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