Immunoblotting procedure

Lasne, F. (2001) Double-blotting a solution to the problem of nonspecific binding of secondary antibodies in immunoblotting procedures. J. Immunol. Methods 253, 125-131. 

In immunoblotting procedures, a mixture of antigens is first separated by gel electrophoresis and subsequently transferred from the gel to a membrane, where it is detected by antibody. Comparative levels of reactivity with antibody can be assessed by incubation with serially diluted antibody preparations. A comprehensive review of the technique is available.44 Because the test is done with proteins that are at least partially degraded, relatively distant relationships can be detected.43 46 The method has been used to calculate SDI values for proteins of potyviruses.47... 

This chapter will describe in detail the procedure for Western blotting of polypeptides and proteins separated on a denaturing polyacrylamide gel system. This procedure is used routinely in our laboratory for the analysis of polypeptides from a variety of subcellular fractions of whole tissue and cell lines, and has evolved over a number of years in the hands of several people. Many different immunoblotting procedures are currendy available details of variadons from the I-protein-A method described here are given in the Notes secdon, as are brief amendments covering electrotransfer from two-dimensional and isoelectric focusing systems. A detailed descripdon of sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) is not appropriate for this chapter, and the reader is referred to vol. 1, Chapter 6, and refs. 9-14. For details of the producdon of polyclonal and monoclonal andsera, Chapters 1-6 in this vol. 

The specificity of fluorescence localization is dependent on the specificity of the primary antibody, a property that must be tested and controlled by other methods, such as immunoprecipitation or immunoblotting. Controls for the labeling procedure described include deletion of the primary antibody step, which controls for the second-step reagent, or inclnsion of a similar, but nonreactive antibody as a first step. In the case of the availability of purified primary antigen, competition controls can be used, but they only control for the reactivity of the antibody with one antigen, and do not mle ont the possibility of a crossreactive, but unrelated antigen. 

Once transfectomas have been generated, they must be screened for the production of the desired fusion. The binding site provided by the expression of the VNP heavy chain in J558L cells and association of the heavy chain with the resident light chain means that the protein fusion can be captured on a solid phase by NP or NIP, and detected using commercially available antibody to mouse X chain and an appropriate enzyme conjugate, in a simple ELISA screening procedure. Similarly, purification can be achieved by affinity adsorption of the fusion onto an NP matrix. An immunoblot method is described here for characterization of selected transfectomas, which allows the mol-wt of the fusion product to be estimated. 

PVDF membranes bind proteins primarily through hydrophobic interactions and are commonly used for their chemical resistance as well as physical stability. High-affinity PVDF membranes such as Trans-Blot (Bio-Rad), ProBlott (Perkin-Elmer), and Immobilon-PSQ (Mil-lipore) are preferred for blots intended for use in N-terminal protein sequencing, whereas low-retention membranes such as Immobilon-P (Millipore) may produce lower background in both immunoblotting and common staining procedures. In addition, low-retention membranes are preferred when proteins will be extracted from the membrane. 

Given the potential of these methods to improve antibody production to multiple antigen mixtures, we performed a comparison of these two methods to a conventional immunization procedure reported previously (6). In addition, we selected two dimensional electrophoresis and immunoblotting as a tool to examine the production of antibodies to minor components of the ECP mixture. 

Figure 4. Separation and detection of ECPs by two dimensional gel electrophoresis. A Silver stained. B Immunoblot with conventional procedure day 112 antisera. C Immuunoblot with cascade procedure day 112 antisera. D Immunoblot with passive immunization procedure day 112 antisera. Exposure time was 24 hours. Reproduced with permission from Ref. 24. Copyright 1989 The Humana Press Inc.

This procedure makes use of electrophoretic and immunochemical techniques to identify a specific protein in a complex mixture. Immunoblotting can be used to (a) determine the presence of a specific protein in a biological preparation (b) determine relative amounts of a specific protein in a biological preparation, and (c) estimate the molecular weight of a specific protein. 

Morsky (1988) has described a method for detecting human lysozyme (with a sensitivity of S5 ng) in body fluids, by a procedure involving immunoblotting. 

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