Production of polyclonal antibodies to maize heat shock proteins

In an effort to avail ourselves of additional tools for studying the response of maize to thermal shifts and/or stresses, some of the maize heat shock polypeptide (HSP) classes have been isolated and purified to produce polyclonal antisera. This is a laborious procedure since the HSPs in maize are synthesized in low amounts and do not accumulate with time as in Drosophila. To purify the HSPs, polypeptide extracts (labelled with 35S-methion1ne) were-electrophoresed on 1-D gels as described previously (Baszczynski et al., 1982). The regions of the gels corresponding to the HSPs were cut out, and the polypeptides were electro-eluted from the gel slices (in a Tris-acetate buffer containing 0.1% SDS) and simultaneously concentrated using an ISCO sample concentrator. The concentrated samples were made 2 mM with respect to PMSF, an aliquot of each sample was re-electrophoresed and purity was checked by fluorographic analysis of the separated polypeptides. This procedure was repeated until high purity was achieved. Four groups of polypeptides were prepared for use as antigens: (a) the 18 kD HSP class, (b) the 73-76 kD class, (c) the 84-89 kD class, and (d) the 73-89 (high molecular weight) class. Alum precipitates of the purified antigens were made. These were emulsified 1:1 with complete Freund's adjuvant and injected subcutaneously into New Zealand White female rabbits. The rabbits were boosted intramuscularly at 6 weeks and at 12 weeks post-injection, and then bled one to two weeks later. The sera were collected, treated at 56 C for 30 minutes to inactivate complement and stored at -20 C until required.

Analysis of the sera by ring tests and Ouchterlony immunodiffusion plates indicated the presence of precipitating antibodies in the sera. Immunoblot analysis of electrophoretically separated polypeptides revealed the presence of antibodies specific to the molecular weight classes of polypeptides against which the antibodies were produced. To screen for the presence of antibodies specific to the maize HSPs (which are detected only by fluorography), total protein extracts from control or heat-shocked seedlings were reacted independently with each of the antisera, the mixtures treated with a cell wall preparation from Staphylococcus aureus to precipitate antigen-IgG complexes, the resulting immunoprecipitates collected and dissolved in lysis buffer, and the samples electrophoresed and analyzed by fluorography.

The results from these studies indicate that: (a) the sera contain antibodies specific to the maize HSPs, (b) there is some cross-reactivity between the various molecular weight classes of HSPs, (c) the high molecular weight HSPs are present in small amounts in non-heat-shocked samples, and (d) there is no detectable synthesis of the low molecular weight 18 kD HSP class in non-heat-shocked material. This suggests that the high molecular weight HSPs exhibit enhanced synthesis following heat shock, while the 18 kD HSP class represent the activation of a set of previously quiescent genes.

The maize HSP antibodies are being used (a) to examine cross-reactivity among different tissues of maize, (b) to compare the immunological similarity in HSPs between different plant and animal species, (c) to purify mRNAs for the HSPs by immunoprecipitation of polysomes from heat-shocked tissues, and (d) for affinity purification of large amounts of HSPs to be used for the production of monoclonal antibodies.

Chris L. Baszczynski
 
 


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