Immobilized E. coli Lysate is a column of beaded agarose to which proteins of an E. coli lysate have been covalently attached. The column and kit are useful for binding and removing (pre-clearing) antibody clones and other components of protein preparations that cross-react with E. coli proteins. This function is especially helpful in reducing non-specific (off-target) binding in Western blotting or ELISA applications with polyclonal antibodies and recombinant proteins expressed in bacteria.

Isolating a recombinant DNA clone that encodes a particular gene or mRNA sequence is accomplished by screening a recombinant DNA library. Once recombinant DNA is synthesized, it is necessary to introduce the hybrid molecule into bacterial cells. Escherichia coli (E. coli) is the usual bacterial host. The resulting library of clones is screened by immobilizing synthesized antigenic material to a solid support and detecting with a sensitive procedure often using an enzyme-labeled secondary antibody. High background, or low signal-to-noise ratio, is often a problem when screening libraries. If the background is localized and unexpected plaques appear to be positive, the primary antibody used may contain components that react with E. coli proteins. Crude antisera and ascites fluid often contain IgG components that bind to E. coli proteins. This could be especially problematic if the titer or binding affinity of the E. coli binding antibodies is higher than that of the antibody to the protein of interest, making the background of the false positive plaques high. Optimizing the dilution of primary antibody for screening may eliminate some of the nonspecific background. By adsorbing the antisera or ascites fluid with an extract of the bacterial strain to inhibit or remove the anti-E. coli IgGs, the nonspecific binding may be further decreased. Another cause of localized background could be cross-reaction of the labeled secondary antibody with bacterial proteins. In this case, it may be beneficial to pre-adsorb the diluted secondary antibody conjugate with an E. coli extract or immobilized support.

To make removal of E. coli antibodies cleaner and easier, Pierce has immobilized E. coli proteins on a solid support. Pierce Immobilized E. coli Lysate consists of partially purified proteins from E. coli strain BMH 71-18 immobilized on agarose. The coupling chemistry is leak-resistant and provides a matrix with low nonspecific binding. A column of Immobilized E. coli Lysate functions as a “reverse” affinity matrix. When a sample is passed over the column, the purified antibody is collected in the void volume. The contaminating E. coli antibodies are absorbed onto the matrix, and the purified antisera is collected.