Application

Antibody/Protein Analysis

The analysis of recombinant monoclonal antibody and protein mixtures for size heterogeneity and purity has widespread applications in biochemistry, molecular biology, and biopharmaceutical laboratories. Typically, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is the technique used for molecular weight-based separations of proteins; however, this technique is slow, labor-intensive, offers marginal quantitation, and is not readily amenable to automation.

Advanced Analytical provides a tool enabling parallel antibody/protein separations by capillary array gel electrophoresis with direct on-line UV absorbance detection (CGE-UV). This rapid, automated method provides improved resolution, reproducibility and quantification when compared to traditional SDS-PAGE methods.

Key Benefits:

• The use of CGE-UV for SDS-protein sizing provides a simple, automated method for protein separation. As most proteins bind SDS in a constant ratio of 1.4 g SDS/g protein, SDS-complexed proteins possess similar mass-to-charge ratios and can be separated according to their molecular weight.
• On-line, direct detection and quantification of protein is performed in UV ranges of 200 or 214 nm, eliminating the need for expensive, complex pre-column derivatization methods or time-consuming stain/destain approaches.
• The parallel system format allows for screening of many different samples in a single experiment, dramatically improving analytical throughput and reducing cost per sample.
• Replaceable gel matrices provide a size-based sieving medium and improve run-to-run reproducibility.
• Detection sensitivity is approximately 5 ng/µL for BSA sampled from 12.5 mM tris-HCl, a level similar to SDS-PAGE Coomassie Blue staining.
• Molecular weight sizing is accurate to within 10% for typical non-glycosylated proteins in the sizing range from 8 kDa to 200 kDa.
• The PRO Size™ software enables automatic extraction of CGE data, calculation of protein molecular weights from comparison to standard markers, relative or absolute protein quantification, and flexible data display and reporting options.

The above results show the CGE-SDS separation of a nine component standard protein mixture using parallel CGE-UV along with a sizing calibration curve. Proteins (in order of MW): ubiquitin (8.6 kDa), lysozyme (14.2 kDa), trypsin inhibitor (21.5 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa), BSA (66.2 kDa), phosphorylase b (97 kDa), β-galactosidase (116 kDa), and myosin (205 kDa).

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