Anti-VAMP8/EDB antibody [EP2629Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

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.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

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.

• IHC-P

PubMed

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

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.

Image from Kamoi M et al., PLoS One. 2012;7(9):e43688. doi: 10.1371/journal.pone.0043688. Epub 2012 Sep 4. Fig 4.; doi:10.1371/journal.pone.0043688; September 4, 2012, PLoS ONE 7(9): e43688.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

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.

• IP

Lab

Immunoprecipitation - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

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.

All lanes:

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

Predicted band size: 11 kDa

Observed band size: 15 kDa

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• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

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).

• WB

Unknown

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

All lanes:

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (ab76021) at 1/20000 dilution

Lane 1:

293T cell lysate at 10 µg

Lane 2:

HeLa cell lysate at 10 µg

Lane 3:

PC12 cell lysate at 10 µg

Secondary

All lanes:

HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

Predicted band size: 11 kDa

Observed band size: 17 kDa

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• WB

Lab

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (ab76021) at 1/10000 dilution

Lane 1:

HEK293 whole cell lysate at 20 µg

Lane 2:

HeLa whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 11 kDa

Observed band size: 15 kDa

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• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (ab76021) at 1/10000 dilution

Lane 1:

Western blot - Human VAMP8 (EDB) knockout HEK-293T cell lysate (<a href='/en-us/products/cell-lysates/human-vamp8-edb-knockout-hek-293t-cell-lysate-ab257791'>ab257791</a>)

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

VAMP8 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human VAMP8 (EDB) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-vamp8-edb-knockout-hek-293t-cell-line-ab266293'>ab266293</a>)

Predicted band size: 11 kDa

Observed band size: 11 kDa

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• WB

Lab

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

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.

All lanes:

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (ab76021) at 1/10000 dilution

Lane 1:

Wild-type A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

VAMP8 knockout A-431 (Human epidermoid carcinoma cell line) whole cell lysate at 20 µg

Lane 2:

Western blot - Human VAMP8 (EDB) knockout A-431 cell line (<a href='/en-us/products/cell-lines/human-vamp8-edb-knockout-a-431-cell-line-ab269584'>ab269584</a>)

Lane 3:

THP-1 (Human monocytic leukemia cell line) whole cell lysate at 20 µg

Lane 4:

SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate at 20 µg

Predicted band size: 11 kDa

Observed band size: 13 kDa

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• WB

Lab

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (AB76021)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-VAMP8/EDB antibody [EP2629Y] (ab76021) at 1/10000 dilution

Lane 1:

Rat kidney tissue lysate at 20 µg

Lane 2:

Mouse kidney tissue lysate at 20 µg

Lane 3:

RAW264.7 whole cell lysate at 20 µg

Lane 4:

NIH/3T3 whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 11 kDa

Observed band size: 15 kDa

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