Quantitative Amino Acid Analysis (AAA)

Quantitative amino acid analysis provides a protein concentration and the molar ration of amino acids present. Our AAA uses PITC derivatization of protein hydrolysates followed by reversed-phase HPLC analysis.

Tryptic Peptide Mapping

In our tryptic peptide analysis, samples are reduced and alkylated then subsequently digested with trypsin. The resulting tryptic peptide mixture is analyzed by reversed-phase HPLC with UV detection.

The tryptic peptide mapping analysis provides a qualitative profile or map of the peptides generated from a trypsin digest of a sample.

GlycoPeptide Mapping

In our GlycoPeptide analysis, samples that have been deglycosylated using the enzyme PNGase F are reduced, alkylated and subsequently digested with trypsin. An additional sample is required for analysis without deglycosylation. The resulting tryptic peptide mixtures are analyzed by reversed-phase HPLC with UV detection. Glycosylated peptides can be identified as peaks that disappear/appear in the peptide maps.

The GlycoPeptide Mapping Analysis provides a qualitative profile or map of the peptides generated from digest of glycosylated and deglycosylated samples. Comparison of the peptide maps aids in determining the number of glycosylation sites in a sample.

Size-Exclusion Chromatography

Size Exclusion HPLC analysis separates proteins based on their hydrodynamic volume. Our assay can detect protein aggregation in a sample down to 1% using SEC-HPLC with UV detection. Our assay can also be used to approximate the molecular weight of a protein sample or can be used to analyze quaternary structure.

Reversed-Phase Chromatography

Reversed-phase HPLC offers a purity analysis that is not dependent upon size or charge as are other common methods for determining purity. RP-HPLC can efficiently separate similar proteins based on hydrophobicity and will provide a quantitative measure of protein impurity in a sample.

Ion Exchange Chromatography

Ion exchange chromatography separates sample components based upon charge differences. It is a quick, robust and quantitative assay to evaluate sample purity and stability. Anion exchange is used to separate species based upon a net negative charge (e.g. sialylation differences, phosphorylation) and cation exchange is used to separate species based upon a net positive charge (e.g. deamidation).

Extinction Coefficient Determination

Extinction coefficients of purified proteins are used to quickly determine the sample concentration by measurement of absorbance at 280 nm. The absorbance of a protein at this wavelength is due mainly to its tryptophan content and to a lesser extent, its tyrosine and cystine content. In order for an extinction coefficient to be accurately determined, a reference sample of known concentration must be used. This can be determined by colorimetric protein assays such as the BCA assay or by quantitative amino acid analysis. Given the concentration of a sample, the extinction coefficient is determined by dividing the measured absorbance by the sample concentration.

SDS-PAGE

SDS-PAGE separates proteins based on size and is used to determine sample purity. Proteins in the polyacrylamide gel are detected using either Coomassie blue or silver stain for more sensitive detection. Our assay provides a measure of sample purity as well as an approximate molecular weight.

IEF

Isoelectric focusing separates proteins based on their charge and can detect heterogeneity in samples due to the presence of post-translational modifications such as glycosylation. The IEF gel can be visualized using either Coomassie blue or silver stain for more sensitive detection. Our assay provides an approximation of the pI of a protein sample as well as an assessment of the extent of heterogeneity in the sample.