Antibody Analysis, Protein Analysis
The analysis of recombinant monoclonal antibody and protein mixtures for size heterogeneity and purity has widespread applications in biochemistry, molecular biology, pharmaceutical, and biopharmaceutical laboratories. Typically, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) is used for molecular weight-based separations of proteins; however, this technique is slow, labor-intensive, offers marginal quantification, and is not readily amenable to automation.
The Protein PRO™ from Advanced Analytical provides an analytical tool for the analysis of antibodies and proteins by parallel protein capillary electrophoresis with direct on-line UV absorbance detection (CGE-UV). This protein capillary electrophoresis system is rapid, automated and 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 and antibody analysis and 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 nm 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 up to 24 different samples in a single experiment. This capability dramatically improving analytical throughput and reducing cost per sample.
- Replaceable gel matrices provide a size-based sieving medium and improved run-to-run reproducibility.
- Detection sensitivity is as low as 2ng/µL, (carbonic anhdyrase), 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 separation and analysis of a commercial antibody under denaturing and reduced conditions and detected on the Protein PRO™ system. Baseline resolution of the nonglycosylated IgG heavy chain fraction is shown next to the IgG heavy chain. In this example 24 separations were conducted simultaneously.
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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