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Development and validation of pharmacokinetic PK and immunogenicity assays supporting demonstration of biosimilarity. Analytical Comparability Studies for Biosimilars Webinar. Furthermore, your purified protein may have a different UV absorption coefficient than the BSA calibration standard. Amino acid analysis — the gold standard for protein concentration determination There are several good reasons why you should use triplicate AAA [2] when you need quantitative data you can trust — for instance to analyze your protein standard solutions: It is a very robust analysis.
It can be performed in the presence of salt and detergents, as it is based on ion-exchange chromatography. The only contaminant that can obstruct the analysis is a chemical containing primary amines. And even then, it might still be possible to do an accurate quantification if the calculations can be based on amino acids not affected by the contaminant. You can compare the results with the sequence. This is a very important feature of the analysis.
By comparing the quantitative data for each amino acid residue with the amino acid sequence of the protein you will get information about the purity of your sample. If the data does not fit within a few residues, your sample is most likely contaminated by other proteins.
It is linear over a broad concentration range. The analysis can measure from Therefore, it is suitable for samples with an unknown protein concentration. Your data can be qualified, using internal and external reference standards.
Even though a majority of biological and biochemical laboratories has, at least at some point, applied one of these methods, the detection and determination of some protein concentrations might not be satisfactory, if possible at all.
Fluorescent labelling of proteins and subsequent detection of the label via fluorescence measurement has proven to be the method of choice in many types of assays. In this case a microplate fluorescence reader is usually the instrument of choice for the measurement.
Although the Pierce assay uses a similar procedure while offering more compatibility in buffer constituents, variability is still a problem compared with biuret-based methods. Consequently, BCA has unofficially become the gold standard among biomedical laboratories for protein measurement preparatory to standard procedures such as Western blotting. Because of differences in chemistry, BCA and Bradford are mutually incompatible in the affinity of one detection reagent over another for particular amino acids.
Proteins with excess cysteine, tyrosine, and tryptophan residues will skew BCA assay absorbances because of their affinity for cuprous ions, while the affinity of Coomassie dye for arginine and lysine will do the same, providing inaccurate readings. Finally, for specialists who may be examining small peptides at low concentration or proteins associated with lipid bilayers that can interfere with standard assay reagents, there are several kits and protocols that allow reliable and reproducible measurement.
One example is the CBQCA assay, designed for highly sensitive measurement of proteins in lipid solutions, which uses potassium cyanide danger! In addition to CBQCA, there is a seemingly limitless variety of specialty applications for protein quantification. The table below provides a summary of differences and compatibilities between the more standard assays.
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