How does the Glycoprotein Carbohydrate Estimation Kit work?
Glycoprotein is oxidized with sodium meta-periodate, which forms aldehydes. When reacted with aldehydes, the proprietary Aldehyde Detection Reagent becomes purple with an absorption maximum at 550nm. The color intensity is proportional to the percentage of carbohydrate (aldehyde) component in the glycoproteins.
Why does this assay estimate rather than quantify carbohydrates?
The results produced by this assay are an approximation of carbohydrate content, because the assay detects sialic acid, galactose and mannose. Therefore, sialic acid-rich glycoproteins will produce a higher absorbance than glycoproteins with a similar percentage of glycosylation but with fewer sialic acid residues.
What is the detection range of this assay?
The assay's estimation range is 3.2% (ovalbumin) to 41.4% (a1-acid glycoprotein). The protein concentration required for the assay is based on the percent carbohydrate (w/w). Highly glycosylated proteins are assayed at 0.25mg/ml. Less glycosylated proteins are assayed at 2.5mg/ml. Researchers with unknown samples should test them at both concentrations to ensure the sample is within the absorbance range for the standard curve.
How many samples can be processed with this kit?
The kit contains sufficient reagents for processing 250 microplate assays or 60 test tube assays and includes negative and positive controls.
Can I measure the absorbance at a wavelength different than 550 nm?
Yes. The purple solution produced when oxidized carbohydrates react with the Aldehyde Detection Reagent can be measured at 530nm and 560nm.
Can I use this kit to estimate free sugars?
Yes. Free sugars form aldehydes when oxidized, which react with the Glycoprotein Detection Reagent.
Can I use this kit to estimate the carbohydrate content of cell lysates or do I need to purify the protein?
The kit is designed for purified proteins but can also estimate the w/w percentage of carbohydrate in total protein.
What is the difference between Glycoprotein Carbohydrate Estimation Kit and the Pierce Glycoprotein Staining Kit (#24562)?
Glycoprotein Carbohydrate Estimation Kit estimates the carbohydrate content of glycoproteins in solution; however, the Pierce Glycoprotein Stain directly stains oxidized glycoprotein on gels and nitrocellulose membranes and does not estimate carbohydrate content.
Will the detergent in my sample interfere with the assay?
Most commonly used detergents do not interfere; however, detergents with sugar derivatives, such as octyl-b-glusoside (OG) and octyl-b-thioglucopyranoside (OTG), can contribute to the signal, resulting in an erroneously high percent carbohydrate value.
Will the reducing agent in my sample interfere with the assay?
Yes. Reducing agents interfere with this assay. The table below lists the compatibility of other commonly used reagents.
|Thermo Scientific Glycoprotein Carbohydrate Estimation Kit reagent compatibility table