Proteasome 20S α4 subunit (human) monoclonal antibody (MCP34)

Aliquot undiluted antibody into smaller volumes (not less than 10μl) prior to freezing if appropriate. The use of high quality ‘antiserum-grade’ plastic or glass vials is recommended. Store diluted antibody at 2-4°C (do not freeze) and use within 1 month. Dilute to working strength with phosphate buffered saline pH 7.2-7.4 and 1% normal goat serum (if a goat anti-mouse IgG linker antibody is to be used) .

Western Blot (1:1,000, colorimetric) Suggested dilutions/conditions may not be available for all applications.Optimal conditions must be determined individually for each application.

The hybridoma secreting the antibody to subunit α4 was generated by fusion of splenocytes from Balb/c mice which had received repeated immunisation with human placenta derived proteasomes. The antibody (clone MCP34) has been extensively characterised by one- and two-dimensional Western blotting and has been demonstrated to immunoprecipitate the native 20S proteasome. Various systems for the nomenclature of the proteasome subunits have been established. This may be a source of confusion as the system on UniProt differs from “standard” nomenclature as described in the literature. The UniProt ID and Gene Name will help to clearly identify the proteins.

Western blot: Single dimension SDS-PAGE of a human placental proteasome preparation or HeLa cell lysate followed by Western blotting gives a single band with a relative molecular weight of approximately 28kDa. Upon 2D analysis antibody PW8120 also reacts with two other proteins with the same molecular weight but differing pI. The amount of protein in the spots decreases with their pI. Peptide mapping suggests that the proteins whilst different are closely related. Proteasome preparations containing degradation products often give reaction patterns with several more spots than that shown. Immunoprecipitation: This antibody has been used for immunoprecipitation using standard procedures using immobilised protein A/G for capture. This antibody is also available immobilized on activated agarose (Prod. No. BML-PW9005) and has been used to precipitate proteasomes from a HeLa cell lysate (Prod. No. BML-SW8750) .