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Western blot analysis of MUC1 monoclonal antibody, clone SM3 (Cat # RAB01094).
MCF-7 cell lysate (35 ug protein in RIPA buffer) were resolved on a 10% SDS PAGE gel and blots probed with the chimeric rabbit antibody (Cat # RAB01094) at 1 ug/mL before detection using an anti-rabbit secondary antibody. A primary incubation of 1h was used and protein was detected by chemiluminescence. The predicted band size for unmodified MUC1 is 122.1kDa, though in breast cancer cell lines like MCF-7 MUC1 can be up to 90% glycosylated (c.f. Mueller et al. PMID: 10373415; T47D cells) and expected band sizes are ~250-300kDa. Thus the two bands likely represent processed (>250kDa) and unprocessed (~121kDa) populations of the protein. Cat # RAB01094 successfully detected human MUC1 in MCF-7 breast cancer cells.

Immunohistochemical staining of human lung tissue using MUC1 monoclonal antibody, clone SM3 (Cat # RAB01094).
Immunohistochemical staining of paraffin embedded human lung tissue using the rabbit-chimeric antibody (Cat # RAB01094). Antigen retreival was acheived by microwaving in citrate buffer (pH 6), followed by blocking with protein block serum-free buffer. Primary antibody incubation with Cat # RAB01094 was carried out at 4 ug/mL for 30 minutes. Samples were then incubated with an anti-rabbit IgG HRP secondary antibody for 20 mins followed by DAB, and counter-staining with haemotoxylin. Staining of type II pneumocytes may be observed. Recommended concentration, 2-4 ug/mL.

Immunofluorescence staining of fixed MCF-7 cells with MUC1 monoclonal antibody, clone SM3 (Cat # RAB01094).
Immunofluorescence analysis of unpermeabilisd paraformaldehyde fixed MCF-7 cells on coverslips stained with the chimeric rabbit antibody (Cat # RAB01094) at 10 ug/mL for 1h followed by Alexa Fluor®488 secondary antibody (1 ug/mL), showing clear membrane staining. The nuclear stain is DAPI (blue). Panels show from left-right, top-bottom; Cat # RAB01094, DAPI, merged channels and an isotype control. The isotype control was stained with unimmunised rabbit IgG followed by Alexa Fluor®488 secondary antibody.

Flow-cytometry using MUC1 monoclonal antibody, clone SM3 (Cat # RAB01094).
MCF-7 cells were stained with unimmunized rabbit IgG antibody (black line) or the rabbit-chimeric antibody (Cat # RAB01094, blue line) at a concentration of 10 ug/mL for 30 mins at RT. After washing, bound antibody was detected using anti-rabbit IgG JK (FITC-conjugate) antibody at 2 ug/mL and cells analyzed on a FACSCanto flow-cytometer.