Biospectroscopy

Protein Concentration by Bradford, BCA, or Lowry

Protein detection and quantification using Bradford, BCA or Lowry Protein quantification Protease Assay Kit is employed. These cuvette or microwell based assays are tested in conjunction with the client’s sample to detect and quantify all levels and forms of protein. Concentration is reported in mass per volume sample. Use of these methods for specific applications will be evaluated for repeatability, linearity, LOD/LOQ and accuracy.

• Sample required: microgram to milligram amounts of protein

Protease Assays

Protease detection and quantification using the Molecular Probes EnzChek Protease Assay Kit is employed (Analysis #6652). This fluorescence, 96-well based assay is tested for its ability to detect and quantify all levels and forms of protease activity. Activity is reported in units per volume sample. Use of this kit for specific applications will be evaluated for repeatability, linearity, LOD/LOQ and accuracy. Also the need to quantify the material using a mass-fluorescence signal calibration curve with the BODIPY dye is examined.

• Sample required: microgram to milligram amounts of protein

Lipase

A method for lipase detection and quantitation using the Molecular Probes Lipase Substrate (green fluorescence) is performed. This fluorescence, 96-well based assay will be tested for its ability to detect and quantify all levels and forms of protease activity. Activity is reported in units per volume sample. The triacylglycerol-based EnzChek lipase substrate offers higher throughput and better sensitivity than chromogenic (TLC or HPLC) assays, and a visible wavelength–detection alternative to pyrene-based fluorescent substrates. In the presence of lipases, the non-fluorescent EnzChek lipase substrate produces a bright, green-fluorescent product (excitation/emission maxima of ~505/515 nm) for the accurate and sensitive detection of lipase activity in solution. Furthermore, the green-fluorescent product of the EnzChek lipase substrate exhibits pH-insensitive spectra in the physiological pH range and is compatible with optics used for fluorescein detection in spectrofluorometers. .

• Sample required: microgram to milligram amounts of protein

Nuclease Assays

A method for nuclease detection and quantification using the Ambion RNase and/or DNase QC kit is performed (Analysis #6727). This fluorescence, 96-well based assay is tested for its ability to detect and quantify all levels and forms of nuclease activity. Activity is reported in units per volume sample. Use of this kit for specific applications will be evaluated for repeatability, linearity, LOD/LOQ and accuracy. Also the need to quantify the material using a mass-fluorescence signal calibration curve with the BODIPY dye is examined.

• Sample required: microgram to milligram amounts of protein

Amylase Assay

A method for amylase detection and quantification is adapted using the Molecular Probes Amylase Assay Kit. This fluorescence, 96-well based assay is tested for its ability to detect and quantify all levels of amylase. Activity is reported in units per volume sample. Use of this kit for specific applications will be evaluated for repeatability, linearity, LOD/LOQ and accuracy. Also the need to quantify the material using a mass-fluorescence signal calibration curve with the BODIPY dye is examined.

• Sample required: microgram to milligram amounts of protein

Extinction Coefficient Determination

The extinction co-efficient is a key analytical parameter from which many assays required the protein concentration. Typically, the client will provide the sequences of the product. The determination is carried out via AAA and the measurement of the absorbance at 280 nm. Beer's law will be used to determine the extinction coefficient.

A Nano-Drop Spectrophotometer or Molecular Devices M2 spectrophotometer are used in determining the absorbance at 280 nm.

Amino Acid Analysis is performed following Analysis #10030. Amino acid analysis is carried out in two steps: 1) acid hydrolysis (usually hydrochloric acid) of protein into component amino acids using the validated Eldex Hydrolysis/Derivatization Workstation; 2) Derivatized the amino acids using the Waters AccQTag Chemistry. Eldex Hydrolysis/Derivatization Workstation Overview: This system is designed to provide convenient hydrolysis and pre-column derivatization as it applies to protein hydrolysis and amino acid modifications performed in the Biopharmaceutical Services area. Typically 6N hydrochloric acid at 110 deg C for 24 h is used to hydrolyze the protein and then it is removed under reduced pressure prior to derivatization. A Waters UPLC is used to separate the derivatized amino acids and from standard plots, the unknown amino acid concentration is quantified.

• Sample required: microgram to milligram amounts of protein