I am observing poor specific signal in my gel stained with Pro-Q Diamond Phosphoprotein Gel Stain, or also observe a weak total protein staining pattern when I switch to a different excitation or emission filter. What is causing this?
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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I have only stained my gel with Pro-Q Diamond Phosphoprotein Gel Stain and am observing the same staining pattern I get with a total protein stain, including seeing all six bands in the PeppermintStick marker lane. A long destain does not improve specificity. What is happening?
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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I have stained my gel with Pro-Q Diamond Phosphoprotein Gel Stain and am observing some staining of non-phosphorylated proteins, including seeing more than two bands in the PeppermintStick marker lane. What can I do to improve phosphoprotein staining specificity?
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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I am observing poor phosphoprotein-specific staining and a high background with Pro-Q Diamond Phosphoprotein Gel Stain. What is happening?
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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Can I use other molecular weight standards with the Pro-Q Diamond Phosphoprotein stain?
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.
