Evaluation of seven oestrogen receptor beta antibodies for immunohistochemistry, western blotting, and flow cytometry in human breast tissue.
Two oestrogen receptors, ER alpha and ER beta, exist. While much is known about ER alpha, the role of ER beta is still undefined, especially at the protein level. The aim of this study was to determine the utility of seven ER beta antibodies (14C8, 8D5, PAI313, PPG5/10, N19, 9.88, and D7N) raised against different domains of ER beta in three commonly used laboratory applications, namely immunohistochemistry, western blot, and flow cytometry, using human breast material. For immunohistochemical analysis of frozen material, PAI313 and D7N gave stronger and more specific signals than 14C8, 8D5, and PPG5/10. In paraffin sections, 14C8, closely followed by PPG5/10, gave by far the most superior nuclear immunoreactivity, compared with the other antibodies tested. In general, flow cytometry results mirrored the immunohistochemistry data for paraffin sections, with antibodies ranked 14C8 > 8D5> or = PAI-313 > PPG5/10 >D7N. For western blotting, 8D5 and D7N yielded the strongest and most consistent bands, with weaker bands seen with the others. It is concluded that ER beta protein can be detected using specific antibodies. However, there is considerable variation between the specificity and application of these antibodies, highlighting the fact that careful optimization is required when selecting an antibody for use in a particular laboratory technique.
Skliris
G P
GP
Molecular Medicine Unit, University of Leeds, Leeds, UK.
Parkes
A T
AT
Limer
J L
JL
Burdall
S E
SE
Carder
P J
PJ
Speirs
V
V
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D014408:Biomarkers, Tumor / Q000032:analysis / Q000276:immunology*; D015153:Blotting, Western / Q000379:methods; D001943:Breast Neoplasms / Q000737:chemistry*; D015925:Cryopreservation; D047629:Estrogen Receptor beta; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007124:Immunoenzyme Techniques; D009363:Neoplasm Proteins / Q000032:analysis / Q000276:immunology; D016612:Paraffin Embedding; D011960:Receptors, Estrogen / Q000032:analysis / Q000276:immunology*
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D047629:Estrogen Receptor beta; D009363:Neoplasm Proteins; D011960:Receptors, Estrogen