Anti-BAP31 antibody [7A3BB6] (ab112993)

Lanes 1-4: Merged signal (red and green). Green - ab112993 observed at 28 kDa. Red - loading control ab52901 observed at  kDa.

 ab112993 Anti-BAP31 antibody [7A3BB6] was shown to specifically react with BAP31 in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266634 (knockout cell lysate ab257857) was used. Wild-type and BAP31 knockout samples were subjected to SDS-PAGE. ab112993 and Anti-beta Tubulin [EP1331Y] - Microtubule Marker (ab52901) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preadsorbed (ab216777) and Goat anti-Mouse IgG H&L (IRDye^® 800CW) preadsorbed (ab216772) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

ab112993 at 5 µg/ml staining BAP31 in Human fibroblast cells by Immunocytochemistry (4% paraformaldehyde fixed and 0.1% Triton X-100 permeabilized) followed by Alexa Fluor^® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1 hour (green). The target locates mainly in the endoplasmic reticulum.

IHC image of BAP31 staining in Human normal colon formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab112993, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.