Recombinant Anti-VAMP8/EDB antibody [EP2629Y] (ab76021)

False colour image of Western blot: Anti-VAMP8/EDB antibody [EP2629Y] staining at 1/10000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab76021 was shown to bind specifically to VAMP8/EDB. A band was observed at 11 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in VAMP8 knockout cell line ab266293 (knockout cell lysate ab257791). To generate this image, wild-type and VAMP8 knockout HEK-293T 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.

Lanes 1 - 4: Merged signal (red and green). Green - ab76021 observed at 13 kDa. Red - loading control, ab7291 (Mouse anti-Alpha Tubulin [DM1A] observed at 55kDa.

ab76021 was shown to react with VAMP8/EDB in wild-type A431 cells in western blot. Loss of signal was observed when VAMP8 knockout sample was used. Wild-type and VAMP8 knockout A431 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab76021 and ab7291 (Mouse anti-Alpha Tubulin [DM1A] overnight at 4°C at a 1 in 10000 Dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of Human lacrimal gland tissue staining VAMP8/EDB with unpurified ab76021.

Antigen retrieval was performed using antigen retrieval solution in a microwave. Sections were blocked with 10 goat serum for 30 minutes and incubated with primary antibody (1/100) overnight at 4°C. Staining was detected using DAB.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue labelling VAMP8/EDB with unpurified ab76021 at a dilution of 1/100. A HRP/AP polymerized secondary antibody was used.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney tissue labelling VAMP8/EDB with purified ab76021 at a dilution of 1/250. Heat mediated antigen retrieval was performed using EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of PC-12 cells labelling VAMP8/EDB with purified ab76021 at a dilution of 1/100. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1: primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

Intracellular Flow Cytometry analysis of HeLa cells labelling VAMP8/EDB with purified ab76021 at a dilution of 1/150 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluorr^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies. 

ab76021 (purified) at 1/50 immunoprecipitating VAMP8/EDB in HEK293 whole cell lysate.

Lane 1 (input): HEK293 whole cell lysate (10µg)

Lane 2 (+): ab76021 + HEK293 whole cell lysate.

Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76021 in HEK293 whole cell lysate.

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.