Detection and quantification of proteins – Western blot

What does blotting paper have in common with bioanalytics? When the surplus ink is absorbed, an identical imprint of the original is created and this is exactly what the “Western blot” also achieves – only not of ink, but of separated individual proteins.

The method was developed in 1979 by J.Renart [1] (and dubbed “Western blot” by W.N. Burnette) and was developed by H. Towbin [2] into the form frequently used today. After transfer of the proteins, they can be stained non-specifically (e.g. using Ponceau red) or specifically (immunodetection). In the latter case, the principle of antigen-antibody binding is used to obtain a detection by means of specific antibody conjugates.

These conjugates are coupled with an enzyme, often horseradish peroxidase or alkaline phosphatase, or a fluorescent dye (e.g. FITC = fluorescein isothiocyanate). Radioactive labelling of the antibody is also possible. If horseradish peroxidase is used, substrate is added, either luminol for a chemiluminescence reaction or 3,3′,5,5′-tetramethylbenzidine, which forms a blue colour complex. If luminol is used, chemiluminescence can be detected by means of a suitable scanner (C-Digit, Li-Cor) and the amount of protein quantified.

 Figure 1: top: SDS-PAGE
            bottom: Measurement of the blot using a C-DiGit blot scanner from Li-Cor.

 Figure 2: Comparison of non-specific and specific protein staining
            a) Staining of the membrane after blotting with Ponceau S
            b) Staining of the membrane with antibodies (1. AK Protein-L-Biotin antibodies, 2 Streptavidin-POD antibodies)

Literature for further study:

[1] Renart, J. et al. (1979): Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure. In: Proc. Natl. Acad. Sci. U. S. A. Volume 76, pp. 3116-3120. PMID 91164.

[2] Harry Towbin, Theophil Staehelin, Julian Gordon: Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. In: Proceedings of the National Academy of Sciences of the United States of America. Volume 76, no. 9, 1979, pp. 4350-4354, PMID 388439, PMC 411572

Obtaining recombinant DNA for cloning – PCR

Based on the basic characteristics of PCR (and also as explained on the Biochemistry pages), this DNA copier is used in analytical biochemistry to produce recombinant DNA for plasmids and to detect successful transformations in combination with agarose gel electrophoresis.

In the former case, a subregion of the DNA is classically amplified from an existing plasmid, the purified PCR product obtained is ligated into a previously cut vector and the new plasmid thus generated is transformed into bacterial cells. A successful transformation can be demonstrated using selection media, but also by means of a renewed PCR. The advantage of PCR is that it can also show whether the desired plasmid and the previously ligated DNA fragment have been taken up by the bacteria without any changes. The PCR can be performed as a so-called colony PCR, in which colonies are “picked” from the overgrown Petri dish with a sterile toothpick and directly added to the reaction mixture. However, the classical route via plasmid extraction with subsequent PCR is also possible. After the PCR, an agarose gel electrophoresis is performed to determine the length of the DNA fragments.

Figure 1: left: PCR device from Bio-Rad
           right: Agarose gel electrophoresis chamber with power supply unit (Bio-Rad)

Figure 2: left: Transilluminator with dark hood for viewing agarose gels
           right: open dark hood with view of transilluminator, fluorescent bands show
                       DNA 

Purification of proteins – LC Äkta

Analytical biochemical methods are also used for the purification of expressed proteins from bacterial cultures and for the analysis of protein mixtures. The low-pressure LC method is the most commonly used method in this context (see also Biochemistry laboratory). In the Analytical biochemistry laboratory, a fully temperature controlled ÄKTA system is available. Depending on the requirements, columns for affinity chromatography (e.g. for His-tag purification), ion exchange (IC), or size exclusion chromatography (=SEC, gel filtration) can be used here.

Other immunoassays – ELISA, immunodiffusion

In addition to the Western blot and other immunoassays already listed, the ELISA (Enzyme-Linked Immunosorbent Assay) and the immunodiffusion method are also used in the Analytical biochemistry laboratory. These methods are also used for the detection or quantification of antigens or antibodies.

A variant of the ELISA is the sandwich ELISA. First, an antibody directed against an epitope of the antigen under investigation is immobilized in a multiwell plate. Then, the antigen to be analysed is added, passed through several washing steps and incubated again with a second antibody, which is also directed against the antigen but another epitope. Due to this structure, the construction resembles a sandwich. The secondary antibody, also called detection antibody, is again radioactively labelled or marked with an enzyme or a fluorescent dye (see “western blot”). The entire system can be automated using suitable equipment. In the Analytical biochemistry laboratory, a washer (Tecan) is used to automate the washing steps before the antigen content is determined with the aid of a plate photometer (Tecan Sunrise).

Different variants are also available for immunodiffusion. In the practical laboratory course in Immunochemistry, the different types are applied and evaluated. Simple radial immune diffusion tests also allow a quantitative statement about existing antigens. However, if the question is: Are rats, cattle, horses, and pigs related to rabbits? At least the immunological relationship between two antigens can be determined by double-radial immunodiffusion according to Ouchterlony. Small holes are punched into a Petri dish containing agar and the antigens to be analysed are pipetted into these holes and the corresponding antibody is pipetted into the middle. The immunological relationship can ultimately be determined from the precipitation lines formed.

 Figure 1: Double-radial immunodiffusion according to Ouchterlony

 Figure 2: Single radial immunodiffusion according to Mancini