Pro-Q™ Emerald 300 Glycoprotein Gel Stain Kit, with SYPRO™ Ruby Protein Gel Stain

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.

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

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.

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

- 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.

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

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.

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