Simultaneous quantitative analysis of multiple protein species within a single sample using standard scanning densitometry

• Published in: Journal of immunological methods Vol. 323; no. 2; pp. 109 - 113
• Main Authors: Bromage, E.S., Kaattari, S.L.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 30.06.2007; Elsevier
• Subjects: Animals; Antibodies - analysis; Antibody; Biological and medical sciences; Blotting, Western; Densitometry; Densitometry - methods; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Molecular immunology; Oncorhynchus mykiss - immunology; Proteins - analysis; Redox; Sensitivity and Specificity; Software; Techniques; Teleostei; Western blotting; Redox; Densitometry; Antibody; Western blotting; Immunoblotting assay; Standards; Protein; Quantitative analysis; Immunological method
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/j.jim.2007.03.007

It is often desirable, when conducting Western blot analyses, to accurately quantify the relative expression of multiple target proteins in a single sample. A common problem occurs: however, when the target proteins vary beyond the linear range of the detection system; thus precluding accurate densitometric analysis for all samples. For example, analysis of teleost immunoglobulin structure under non-reducing but denaturing conditions, yields multiple, differentially polymerized forms (redox forms) within a single sample, which can exceed single log differences in concentration, as visualized by chemiluminescent and X-ray film development. To resolve this difficulty an efficient technique has been developed that uses dilutions of a single sample, allowing accurate quantification of target proteins within their potentially unique and varied linear range of detection. Upon consideration of the respective dilution factor that yields an appropriate estimate, the multiple targets can be quantified. When the results from this technique are compared to other systems possessing more expansive linear ranges, the results obtained are comparable to within 1%. Thus, laboratories without access to more sensitive and costly densitometric instrumentation can still employ standard densitometric analysis to accurately quantify multiple targets in a single sample.