Recombinant Anti-GAS 6 antibody [EPR23931-27] (ab264098)

False colour image of Western blot: Anti-GAS 6 antibody [EPR23931-27] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab264098 was shown to bind specifically to GAS 6. A band was observed at 75 kDa in wild-type HEK-293 cell lysates with no signal observed at this size in GAS6 knockout cell line ab275837 (knockout cell lysate ab275811). To generate this image, wild-type and GAS6 knockout HEK-293 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized THP-1 (Human monocytic leukemia monocyte) cells labelling GAS 6 with ab264098 at 1/500 dilution (Right) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Left). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were stained with rabbit IgG or ab264098.

Blocking and diluting buffer and concentration: 5% NFDM/TBST

Negative control: Jurkat (PMID:10400186)

Samples are non-boiled as boiling may cause protein aggregates.

Exposure time: Lanes 1-2: 48 seconds;Lanes 3-4: 3 minutes.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized THP-1 (Human monocytic leukemia monocyte) cells labelling GAS 6 with ab264098 at 1/500 dilution (0.1ug)/ Right (Red) compared with a Rabbit monoclonal IgG (ab172730) / Left isotype control. A Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Cells were stained with rabbit IgG or ab264098. Cells were co-stained with DRAQ/ Ki-67 to differentiate cell cycle phase.

GAS 6 was immunoprecipitated from 0.35 mg THP-1 (human monocytic leukemia monocyte) whole cell lysate with ab264098 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab264098 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: THP-1 (human monocytic leukemia monocyte) whole cell lysate 10 ug

Lane 2: ab264098 IP in THP-1 whole cell lysate

Lane 3: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated: Rabbit monoclonal IgG (ab172730) instead of ab264098 in THP-1 whole cell lysate

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 15 seconds

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized Jurkat (Human T cell leukemia T lymphocyte (Left) / THP-1 (Human monocytic leukemia monocyte, Right) cells labelling GAS 6 with ab264098 at 1/500 dilution (Right). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody. Negative control: Jurkat (PMID:10400186)