Dimethyl pimelimidate

Dimethyl pimelimidate (DMP) is a homobifunctional crosslinking agent that has imidoester groups on either end (Thermo Fisher). The imidoesters are amine reactive to give stable amidine linkages with target molecules. The 7-atom bridge created by DMP crosslinks is non-cleavable and... 

In addition, antibodies may also be coupled to other previously immobilized proteins. For example, the antibody may be first captured on protein A or protein G media and then cross-linked to the immobilized protein A or G with reagents such as glutaraldehyde or dimethyl pimelimidate. The advantage here is that the antibody need not be pure prior to coupling because the protein A or protein G will selectively bind only antibody and none of the other serum proteins. The disadvantage is that free protein A or protein G will still be available to cross-react with any free antibody in samples to be analyzed, which will only be problematic with serum-based samples. 

Most MAbs or polyclonal antibodies used in immunodetection procedures are either mouse or rabbit antibodies. With the exception of mouse IgG subclass 1, all these antibodies can be coupled to protein A-sepharose, allowing maximal correct orientation of the antibody for immunoprecipitation. The use of the CTOSslinker dimethyl pimelimidate (15) also means that the antibody remains attached to the protein A beads during electrophoresis producing cleaner Western blots. 

Protein A/G-Ig complex can be stabilized with the bifunctional crosslinker dimethyl pimelimidate (24, 25). In this form the antibody on the column is oriented with the Fab arms free from the matrix and available for antigen binding Protocol 4). Complete kits for immobilization of antibody on Protein A or G are marketed by Pierce (IgG Orientation Kit), but any source of Protein A-or Protein G-agarose will do. The differences between Proteins A and G lie in the specificity for different immimoglobulin isotypes and species. Protein G in particular binds to rat IgG isotypes that Protein A does not. The majority of monoclonal antibodies made by conventional ceU fusion will be IgGs of mouse or rat origin, so Table 2 shows the relative affinities of these two proteins for different rodent IgG isotypes. 

Dimethyl pimelimidate dihydrochloride (Pierce, 21666), dissolved at 25 mM in crosslinking buffer... 

Figure 2. Fluorohydrolase activity as a function of pH for dimethyl pimelimidate crosslinked CCMP. Activity was measured using 5 mM PMSF as the substrate at 30°C. Each activity is the average of two separate measurements obtained by HPLC and fluoride electrode methods described in the text. 

Conformationally modified RNase, derivatized with dimethyl pimelimi-date, had the highest fluorohydrolase activity toward DFP however, the highest activity toward PMSF was obtained for dimethyl suberimidate crosslinked protein. The best overall activity for both substrates was obtained with the pimelimidate (Cs) crosslinked modified RNase. A plot of activity towards PMSF versus pH, after chromatography on G-15 Sephadex, shows that the optimum pH is approximately 7.5 (Figure 2). The values of... 

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