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

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.

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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.

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