Pro-Q™ Diamond Phosphoprotein Gel Stain, 1 L - FAQs

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.

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

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.

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

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.

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

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.

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

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.

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

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

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

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.

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

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.