Pro-Q™ Diamond Phosphoprotein Gel Stain, 200 mL - FAQs

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 Diamond信号淬灭。如果您在使用Pro-Q Diamond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。

这表示Pro-Q Diamond染色剂已降解，应丢弃染色液。可能是因为染色剂已超过保质期或在保存期间过多暴露于室内灯光。暴露于室内灯光会使染色剂分子逐渐降解，切割磷酸盐结合部分，将染色剂变为非特异性蛋白染色剂。这会发生在染色剂光漂白之前，但是整体信号会比非降解染色剂的特异性信号弱。染色后的凝胶不太可能被保存，但您可尝试通过重复过夜固定步骤而完全去除染色剂，通过水洗去除固定剂，然后使用新的Pro-Q Diamond磷酸化蛋白凝胶染色剂再次染色。

为获得最佳染色特异性，应除去凝胶上的所有SDS和固定剂。固定步骤可除去SDS，水洗步骤可除去固定剂。为确保除去所有SDS和固定剂，有必要进行多次固定及多次水洗。在固定和水洗步骤中，较大的或较厚的凝胶可能需要更多的溶液或更长的孵育时间，或者也可以进行微波染色。可能需要延长凝胶的脱色时间。将凝胶放回到脱色液中继续孵育，直到PeppermintStick标准品泳道中只能看到2条条带。

为了使Pro-Q Diamond磷酸化蛋白凝胶染色剂正常工作，应在电泳前按着说明书中步骤进行氯仿/甲醇沉淀操作，将样品进行去脂化和脱盐处理。Pro-Q Diamond染色剂也能与磷脂结合，而反离子和高盐浓度可遮盖染色剂与磷酸盐的电荷相互作用。

其他已知的磷酸化蛋白质可以用作Pro-Q Diamond磷酸化蛋白质染料的阳性对照标准品。蛋白质分子量标准品试剂（货号P6649）中的卵清蛋白就是一种磷酸化蛋白质。Mark12、Invitrogen Sharp、SeeBlue或SeeBlue Plus2标准品中的蛋白质，都不是可以用作Pro-Q Diamond磷酸化蛋白质染料的阳性对照的磷酸化蛋白质。

可以。我们推荐在使用Pro-Q Diamond磷酸化蛋白质印迹膜染料染色后，再使用SYPRO Ruby印迹膜染料染色。

不能，Pro-Q Diamond磷酸化蛋白质凝胶染料和印迹膜染料的配方非常不同，交换使用将产生不可接受的磷酸化蛋白检测结果。

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。

Many total protein stains including SYPRO Ruby Gel Stain and Coomassie Blue stain will quench the Pro-Q Diamond signal. If you are staining your gels or blots with Pro-Q Diamond 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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This indicates that the Pro-Q Diamond dye has degraded and the staining solution should be discarded. Either the stain has been used past the stability period or it has been exposed to excessive room light during storage. Exposure to room light will gradually degrade the dye molecule, cleaving the phosphate-binding moiety and turning the dye into a non-specific protein stain. This will happen before the dye photobleaches, although the overall signal should be weaker than the specific signal obtained with non-degraded dye. It is likely not possible to save the stained gel, but you could try completely removing the dye by repeating the fixation step overnight, washing in water to remove fixative and then re-staining using a new stock of Pro-Q Diamond Phosphoprotein Gel Stain.

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All SDS and fixative must be removed from the gel for optimal staining specificity. The fixation step removes the SDS and the water washes remove the fixative. To make sure that all the SDS and fixative are removed, it is necessary to do multiple changes in fixative solution followed by multiple changes in water. Larger or thicker gels may require increased volumes or incubation times in the fixative and water wash solutions, or the microwave staining procedure can be performed. The gel may need a longer time in destain solution. Return the gel to the destain solution and continue to incubate in destain solution until only two bands are visible in the PeppermintStick standard lane.

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For Pro-Q Diamond Phosphoprotein Gel Stain to work properly, it is necessary to delipidate and desalt the sample prior to electrophoresis by following the chloroform/methanol precipitation procedure in the protocol. The Pro-Q Diamond dye will also bind phospholipids and the dye charge interaction with phosphates can be masked by the presence of counter ions and a high salt concentration.

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Other known phosphoproteins can be used as positive control standards for the Pro-Q Diamond Phosphoprotein stain. Ovalbumin, in the Protein Molecular Weight Standards Reagent (Cat. No. P6649) is a phosphoprotein. None of the proteins in the Mark12, Invitrogen Sharp, SeeBlue or SeeBlue Plus2 standards is a phosphoprotein that could be used as a positive control with the Pro-Q Diamond Phosphoprotein stain.

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Yes. We recommend staining with SYPRO Ruby Blot Stain after staining with Pro-Q Diamond Phosphoprotein Blot Stain.

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No, the Pro-Q Diamond Phosphoprotein Gel Stain and Blot Stain are very different formulations and will not give acceptable phosphoprotein detection results on the alternate format.

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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 water 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.

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