Pro-Q™ Emerald 300 Glycoprotein Gel and Blot Stain Kit - FAQs

使用Pro-Q Emerald糖蛋白凝胶染色剂对Invitrogen NuPAGE Bis-Tris和Tris-Acetate凝胶染色时，可能会得到高背景染色，特别是与MES电泳缓冲液结合使用或使用接近有效期限的凝胶时[临近过期的凝胶时]。凝胶背景会随丙烯酰胺密度的增加而增加，梯度凝胶从上到下的背景逐渐增强，与丙烯酰胺梯度相对应。增加洗涤次数或改进孵育时间将有助于降低背景。使用Tris-甘氨酸或Tricine凝胶，会得到更好的结果。如果想继续使用Pro-Q Emerald染色剂对Invitrogen NuPAGE Bis-Tris凝胶进行染色，我们建议使用近期购买的凝胶和MOPS电泳缓冲液。在高背景的凝胶中，仍然能够检测到糖蛋白，但灵敏度降低。

Pro-Q Emerald染色剂存放时间越久，染色剂斑点越多，特别是Pro-Q Emerald 300染色剂，这是因为染色剂会随时间而发生自聚集。染色剂与染色剂容器、溶液中的污染物或来自空气或手套的颗粒物结合，也会形成斑点。灰尘、头发、手套粉末或掉落在凝胶或玻璃成像板上的衣服线头中的自发荧光颗粒物，在成像时也会形成非染色剂斑点。成像仪对凝胶表面特征的聚焦越好，形成的可见斑点越多。

为了将斑点形成和其他背景碎片数量降至最低，应保持实验室整洁卫生，使用电阻率大于18 megohm/cm的超纯水制备溶液，接触凝胶前用水冲洗掉手套上的粉末，使用无尘擦布并穿上实验服或者不要穿可产生大量线头的衣服，每次使用时都用乙醇冲洗染色容器并在下次使用前擦掉所有残留染色剂，每次放置凝胶前用乙醇和水冲洗和擦净玻璃成像表面。在染色和洗涤步骤之间，用乙醇擦掉染色托盘表面积累的染色剂。斑点沉积在凝胶表面后，无法将其洗掉。当分析接近检测限的大量糖蛋白时，我们建议将样品放在凝胶的中间泳道进行电泳。

在凝胶3D图像中，斑点会呈现为较高的尖峰。这些斑点与蛋白质斑点或条带的3D图像看起来不同。一些图像分析软件包具有降噪算法，可轻松识别和去除这类像素化噪音。

许多总蛋白染色剂，包括SYPRO Ruby凝胶染色剂和Coomassie Blue染色剂，会使Pro-Q Emerald信号淬灭。如果您在使用Pro-Q Emerald染色剂对凝胶或印迹膜进行染色时，使用了之前用于总蛋白染色剂的容器，则染色托盘中残留的总蛋白染色剂可能会污染您的凝胶。染色时应使用新的容器（如塑料称量船），每种染色剂使用指定容器，或用乙醇彻底冲洗容器并用Kimwipe纸巾擦去所有残留物。

来自Tris或甘氨酸的伯胺，可导致染色结果较差，并且会与高碘酸盐氧化生成的醛基/酮基反应。这有效覆盖了反应基团，使Pro-Q Emerald染色剂无可结合的活性位点。为了除去所有胺污染物，应增加新鲜固定液的孵育体积或孵育次数，并增加洗涤缓冲液的体积或洗涤次数。

未充分去除高碘酸氧化溶液也可能导致染色结果较差。为了充分去除高碘酸，应在氧化步骤后增加洗涤液体积或洗涤次数。

糖蛋白糖基的氧化不充分也可能导致染色结果较差。应增加高碘酸氧化溶液的体积。

注意：对于小型凝胶，高碘酸氧化的次数或孵育时间不可超过30分钟。过多的高碘酸氧化可导致非糖基化蛋白的染色增加。

使用Pro-Q Emerald糖蛋白凝胶染色剂进行染色时，为获得最佳的染色信号和灵敏度，大型凝胶、非常厚或非常高比例的丙烯酰胺凝胶通常需要更多的孵育或洗涤步骤。

如果CandyCane标准品和待测糖蛋白被正确染色，则表示试剂盒能正常工作。一些丰度非常高的蛋白，如血清和血浆中的白蛋白，会被轻微染色。Pro-Q Emerald染色剂与糖基共价结合，所以，染色后多洗涤有助于去除非共价结合染色剂。在Pro-Q Emerald染色剂然后后再使用总蛋白染色剂（如SYPRO Ruby蛋白质凝胶染色剂）进行染色，可检测高丰度所致的非特异性染色。也就是说，一些蛋白质实际上在天然状态下被大量氧化，而这种羰基化会被Pro-Q Emerald试剂染色。无氧化步骤的Pro-Q Emerald染色，能够区分氨基酸的羰基化与糖基化。在这些条件下，CandyCane标记物泳道不会被染色，但羰基化蛋白会被染色。

在步骤2.5氧化溶液孵育中，使用高碘酸盐过度氧化会导致非糖基化蛋白发生较强的非特异性染色。标准小型凝胶或印迹膜的孵育时间不要超过30分钟，大型2D凝胶不要超过1小时，或者不要使用超过推荐浓度的高碘酸盐。

可以。我们推荐在使用Pro-Q Emerald 300或488糖蛋白印迹膜染料染色后，再使用SYPRO Ruby印迹膜染料染色。

使用Pro-Q Emerald 300/488糖蛋白染料时，必须使用PVDF膜。硝化纤维素膜会被固定步骤中的高浓度甲醇溶解。

最好将凝胶在固定步骤保存过夜，因为甲醇和乙酸都能够沉淀蛋白质并防止扩散。若将容器良好密封以防止污染或凝胶干燥，并且静置或轻轻摇动容器以尽量减少凝胶损害，则凝胶可在固定溶液中长久稳定保存。较高的甲醇浓度会使凝胶脱水、萎缩，并可能变为不透明的白色。这属于正常情况。只需将凝胶置于洗涤液中轻轻摇动，即可使其恢复原状。

也可在固定步骤后将凝胶置于洗涤液中保存过夜。经过固定后洗涤，最好遵循推荐的实验方案时间来完成染色步骤。凝胶染色后，只要是在避光条件下保存，信号可至少维持数天。染色并干燥的印迹膜可收集归档，在避光条件下保存的印迹膜，其信号可一直被检测到。

其他已知的糖蛋白可以用作Pro-Q Emerald 300/488糖蛋白染料的阳性对照标准品。蛋白质分子量标准品试剂（货号P6649）中的卵清蛋白和转铁蛋白都是糖蛋白。Mark12、Invitrogen Sharp、SeeBlue或SeeBlue Plus2标准品中的蛋白质，都不是可以用作Pro-Q Emerald 300/488糖蛋白染料的阳性对照的糖蛋白。

使用Pro-Q Emerald 300/488糖蛋白染料染色前，必须清除蛋白质中的SDS，这是为了使糖残基能够接近氧化试剂（高碘酸）和Pro-Q Emerald染料试剂。SDS可覆盖蛋白质，可能产生空间位阻而限制蛋白质接近试剂。

使用Pro-Q Emerald 300/488糖蛋白染料染色前，必须清除凝胶上所有残留的高碘酸，这是为了限制Pro-Q Emerald染料的氧化/分解。

如果凝胶缓冲液中含有Tris、甘氨酸或任何其他结构中含有伯胺的成分，其中的伯胺会直接与高碘酸氧化形成的醛/酮发生反应，覆盖所有反应位点，使Pro-Q Emerald染料试剂没有结合位点。

Pro-Q Emerald 300/488糖蛋白凝胶染料可以兼容质谱。在胰蛋白酶消化中，只有少数多肽会发生糖基化，而大部分非糖基化的多肽能够被鉴定。糖基化多肽在标准条件下无法被检测到（染色或未染色），因为数据库未将连接在多肽上的糖链考虑在内。

Pro-Q Emerald糖蛋白凝胶染料对Tris-甘氨酸和Tricine凝胶的染色效果最好。Pro-Q Emerald染料可用于Invitrogen NuPAGE Bis-Tris和Tris-Acetate凝胶，但可能产生较高的背景染色，特别是与MES电泳缓冲液一起使用或用于邻近有效期的凝胶时。如果您想将Pro-Q Emerald染料用于Invitrogen NuPAGE Bis-Tris凝胶，我们推荐使用近期购买的凝胶和MOPS电泳缓冲液。凝胶背景随丙烯酰胺密度的增加而增加，梯度凝胶的背景从上到下逐渐减弱，与凝胶上丙烯酰胺的梯度保持一致。在高背景凝胶上，仍然可检测到糖蛋白，但灵敏度降低。

高碘酸氧化试剂通过形成环状高碘酸酯而破坏糖残基上邻近氢氧根（1,2二醇）间的键， 形成醛或羰基基团。Pro-Q Emerald试剂含有伯胺，可与醛/酮直接结合，从而在糖残基和染料分子间形成共价键。

注意：Pro-Q Emerald的精确结构拥有专利权。

Pro-Q Emerald Glycoprotein Gel Stain may show high background staining in Invitrogen NuPAGE Bis-Tris and Tris-Acetate gels, especially in combination with MES running buffer or in gels that are nearing their expiration date. The gel background increases with acrylamide density and gradient gels will show a gradual increase in background from the top to the bottom of the gel corresponding to the acrylamide gradient. Increasing the number of washes or modifying incubation times will not help to reduce this background. Better results will be obtained with Tris-Glycine or Tricine gels. If you wish to continue using Pro-Q Emerald stain with Invitrogen NuPAGE Bis-Tris gels, we recommend using recently purchased gels and MOPS running buffer. Glycoproteins will still be detected in gels with high background, but with reduced sensitivity.

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

Speckling of Pro-Q Emerald dye, especially with Pro-Q Emerald 300 stain, can occur as the Pro-Q Emerald dye ages, due to self-aggregation of the dye over time. Speckles can also form due to dye binding to contaminants from the staining container, solutions, or particles from the air or gloves. Non-dye speckles can also show up in the image from auto-fluorescent particles of dust, hair, glove powder, or clothing lint that falls on the gel or surface of the glass imaging plate. The better the imager is at focusing on surface features of the gel, the more speckles that are going to be visible.

To minimize the formation of speckles and other background debris, follow clean laboratory practices, use ultrapure water of >18 megohm-cm resistance to prepare solutions, rinse gloves in water to remove powder residue before touching gels, use lint-free wipes and wear a lab coat or avoid wearing clothing that generates a lot of lint, always rinse the staining container with ethanol and wipe out any residual dye before staining another gel, and always rinse and wipe down the glass imaging surface with ethanol and water before placing your gel down. Once speckles have been deposited on the gel, it is not possible to wash them off. When analyzing amounts of glycoprotein near the limit of detection, we advise running samples in the middle lanes of the gel.

Speckles will show up as sharp, tall spikes on 3D renditions of gel images. These spikes look distinct from 3D renditions of protein spots or bands. Some image analysis software packages have de-speckling algorithms that can easily identify and remove this type of pixelated noise.

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Many total protein stains including SYPRO Ruby Gel Stain and Coomassie Blue stain will quench the Pro-Q Emerald signal. If you are staining your gels or blots with Pro-Q Emerald stain in containers that have previously been used for a total protein stain, you may be contaminating your gel with residue left on the staining dish from the total protein stain. Either use new containers, such as plastic weigh boats, designated containers for each stain, or rinse the container well in ethanol and wipe out any residual residue with a Kimwipe tissue.

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- Poor staining can be caused by the presence of primary amines, such as from Tris or glycine, that will also react with the aldehyde/ketone groups generated by periodate oxidation. This effectively caps the reactive groups, leaving no reactive sites for Pro-Q Emerald dye to bind. To remove any amine contamination, increase the volume or number of incubations in fresh fixative and then increase the volume or number of washes in wash buffer.
- Poor staining can also be due to inadequate removal of the periodic acid oxidation solution. Increase the volume or number of washes after the oxidation step to ensure adequate removal of periodic acid.
- Poor staining can also be due to inadequate oxidation of glycoprotein sugars. Increase the volume of periodic acid oxidation solution.

Note: Do not increase the number or incubation time of the periodic acid oxidation in excess of 30 minutes for small-format gels. Excessive periodic acid oxidation could result in increased staining of non-glycosylated proteins. In general, large format, unusually thick or very high percent acrylamide gels may require additional incubations or wash steps for optimal signal and sensitivity of staining with Pro-Q Emerald Glycoprotein Gel Stain.

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If the CandyCane standard and test glycoproteins are staining correctly, then the kit is performing well. Some very highly abundant proteins, such as albumin in serum and plasma, may stain lightly. The Pro-Q Emerald dye is covalently attached to sugar residues, so more post-staining washes will help to remove any non-covalently bound dye. Non-specific staining due to high abundance can be determined by post-staining with a total protein stain, such as SYPRO Ruby Protein Gel Stain. That being said, some proteins are actually heavily oxidized in the native state, and this carbonylation will be picked up by the Pro-Q Emerald reagent. Carbonylation of amino acids can be distinguished from glycosylation by performing the Pro-Q Emerald staining without the oxidation step. Under these conditions, the CandyCane marker bands will not be stained, but the carbonylated proteins will.

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Over-oxidation with periodate during the step 2.5 Oxidizing Solution incubation will cause strong non-specific staining of non-glycosylated proteins. Do not incubate standard mini-gels or blots longer than 30 min, or large format 2D gels longer than one hour or use more than the recommended concentration of periodate.

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Yes. We recommend staining with SYPRO Ruby Blot Stain after staining with Pro-Q Emerald 300 or 488 Glycoprotein Blot Stain.

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PVDF membranes must be used with Pro-Q Emerald 300/488 Glycoprotein stains. Nitrocellulose membranes will be dissolved by the high methanol in the fixation step.

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The best step for leaving the gels overnight is during the fixation step, as the methanol and acetic acid both precipitate proteins and prevent diffusion. Gels are stable indefinitely in the fixation solution as long as the containers are well sealed to prevent contamination or gel drying and the containers are allowed to sit or rock gently to minimize gel damage. The high methanol concentration will dehydrate the gel, shrinking it and possibly giving it an opaque, white appearance. This is normal. Simply gently rock the gel in the wash solution to rehydrate to its original appearance.

Gels can also be left overnight in the acetic acid wash after the fixation step. After the post-fix wash, it is best to complete the staining procedure following the recommended protocol times. Once the gels are stained, the signal should be visible for at least several days, as long as the gels are protected from light. Stained and dried blots can be archived and the signal detected indefinitely, as long as the blots are protected from light.

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Other known glycoproteins can be used as positive control standards for the Pro-Q Emerald 300/488 Glycoprotein stains. Ovalbumin and transferrin in the Protein Molecular Weight Standards Reagent (Cat. No. P6649) are glycoproteins. None of the proteins in the Mark12, Invitrogen Sharp, SeeBlue or SeeBlue Plus2 standards is a glycoprotein that could be used as a positive control with the Pro-Q Emerald 300/488 Glycoprotein stains.

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SDS must be removed from the proteins prior to staining with Pro-Q Emerald 300/488 Glycoprotein stains so as to make the sugar residues accessible to the oxidizing reagent (periodic acid) and the Pro-Q Emerald dye reagent. SDS coats proteins and may limit access of the kit reagents by simple steric hindrance.

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Residual periodic acid must be removed from the gel prior to staining with Pro-Q Emerald 300/488 Glycoprotein stains so as to limit oxidation/decomposition of the Pro-Q Emerald dye.

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If the gel buffer contains Tris, glycine or any other component that has a primary amine in its structure, the primary amine will react directly with the aldehydes/ketones formed upon periodic acid oxidation, effectively capping all reactive sites and leaving no sites for the Pro-Q Emerald dye reagent to bind to.

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Pro-Q Emerald 300/488 Glycoprotein Gel Stains are compatible with mass spectrometry. Only a minority of peptides in a trypsin digest will be glycosylated and the majority of non-glycosylated peptides can be identified. Glycosylated peptides are not detected under standard conditions (stained or unstained), as the databases do not take into account the glycans attached. One can deglycosylate the protein, which removes the covalently-bound dye and renders the peptide identifiable.

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Pro-Q Emerald Glycoprotein gel stains work best with Tris-Glycine and Tricine gels. Pro-Q Emerald stains can be used with Invitrogen NuPAGE Bis-Tris and Tris-Acetate gels, but may result in higher background staining, especially in combination with MES running buffer or in gels that are nearing their expiration date. If you wish to use Pro-Q Emerald stains with Invitrogen NuPAGE Bis-Tris gels, we recommend using recently purchased gels and MOPS running buffer. The gel background increases with acrylamide density and gradient gels will show a gradual increase in background from the top to the bottom of the gel corresponding to the acrylamide gradient. Glycoproteins will still be detected in gels with high background, but with reduced sensitivity.

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

The periodic acid oxidizing reagent breaks the bonds between vicinal hydroxyls (1,2 diols) on sugar residues via the formation of a cyclic periodate ester, forming either an aldehyde or ketone carbonyl group. The Pro-Q Emerald reagent contains a primary amine that will bind directly to the aldehyde/ketone, thus forming a covalent bond between the sugar residue and the dye molecule. Note: The exact structure of the Pro-Q Emerald reagents is proprietary.

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