Pierce™ 6xHis Protein Tag Stain Reagent Set - FAQs

这可能是由于含组氨酸簇的蛋白发生了较弱的交叉反应染色。以下是我们的建议：

•延长凝胶水洗时间（第5步）
•稍微缩短染色时间（第2步）
•为了将较弱的非特异性染色降至最低，应调整曝光时间和其他设置。

以下是可能原因和解决方案：

- 紫外光源质量差或曝光不充分：：尽可能使用CCD照相机进行检测；确保紫外灯以合适的波长进行激发（280–310 nm）。
- 目的蛋白表达较差（上样量不足）：每条泳道中电泳的蛋白量不满足检测方法的要求。
- 洗涤不充分；凝胶中残留的SDS阻止染色剂的结合：用超纯水洗涤凝胶3 × 20分钟，然后重新染色。
- 目的蛋白分子量较小（< 20kDa），在洗涤时从凝胶中扩散出来：水洗前，使用50%甲醇：7%乙酸溶液固定凝胶15分钟。
- 染色剂向凝胶中的扩散效果较差：将染色时间延长至10分钟（第2步）；可在同一块凝胶上重复该步骤。

将凝胶保存在水中，荧光信号可稳定维持数小时。保存过夜后，信号仍可检测到，但多少会有所衰减。

Thermo Scientific 6xHis蛋白质标签染色试剂套装的染色步骤中无固定步骤。因此，使用该试剂盒染色不会限制后续使用通用蛋白质染料进行总蛋白质染色或将蛋白质转印到膜上。

注意：在MOPS或MES缓冲液中电泳的Bis-Tris凝胶可能需要在染色步骤前使用50%甲醇：7%乙酸固定15分钟。电泳后，固定凝胶，然后开始染色操作的步骤1。

使用CCD相机，Thermo Scientific 6xHis蛋白质标签染色试剂套装可检测到一个条带中低至0.2 μg的35 kDa (5.7 pmol) His标签蛋白；使用紫外透照仪，该套装可检测到一个条带中低至2 μg (57 pmol) 的His标签蛋白。检测时，需要使用波长范围为280–310 nm的紫外光照射染色凝胶。

Thermo Scientific 6xHis蛋白质标签染色试剂套装可在室温下稳定保存1年。

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.

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

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.