Anti-Giantin antibody - Golgi Marker (ab80864)

Lanes 1 - 2: Merged signal (red and green). Green - ab80864 observed at 377kDa. Red - loading control, ab130007, observed at 130kDa.

ab80864 was shown to recognize GOLGB1 in wild-type HAP1 cells as signal was lost at the expected MW in GOLGB1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GOLGB1 knockout samples were subjected to SDS-PAGE. Ab80864 and ab130007 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 1/1000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

ab80864 stained in Hela cells. Cells were fixed with 100% methanol (5min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab80864 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

IHC image of ab80864 staining in human cervix carcinoma formalin fixed paraffin embedded tissue section*, performed on a Leica Bond^TM 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 ab80864, 0.1µg/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.

*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

Ab80864 staining Giantin in WIF-B cells (hepatoma-derived hybrid cell line) by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with formaldehyde; permeabilized with 0.2% Triton X-100 and blocked with 1% Donkey serum in 0.1% PBST for 1 hour at 22°C. Samples were incubated at 1/25 dilution for 3 hours at 22°C. An Alexa Fluor^® 594 Donkey anti rabbit was used as the secondary antibody at 1/200 dilution.

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