Anti-Nectin 2 antibody [EPR6717]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Nectin 2 antibody [EPR6717] (AB135246)

Immunocytochemistry analysis of HT-29 (Human colorectal adenocarcinoma epithelial cell) cells labeling Nectin 2 with Purified ab135246 at 1 : 50 dilution (3.06 µg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1 : 1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Nectin 2 antibody [EPR6717] (AB135246)

Intracellular Flow Cytometry analysis of HUVEC (Human umbilical vein endothelial cell) cells labeling Nectin 2 with Purified ab135246 at 1/20 dilution (10μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IP

Lab

Immunoprecipitation - Anti-Nectin 2 antibody [EPR6717] (AB135246)

Purified ab135246 at 1/20 dilution (1μg) immunoprecipitating Nectin 2 in SW480 whole cell lysate.
Lane 1 (input) : SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate 10μg
Lane 2 (+) : ab135246 + SW480 whole cell lysate.
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab135246 in SW480 whole cell lysate.
VeriBlot for IP Detection Reagent (HRP) (ab131366) (1/1000 dilution) was used for Western blotting.
Blocking Buffer and concentration : 5% NFDM/TBST.
Diluting buffer and concentration : 5% NFDM/TBST.
Observed band size : 70-85 kDa

All lanes:

Immunoprecipitation - Anti-Nectin 2 antibody [EPR6717] (ab135246)

Predicted band size: 58 kDa

false

• WB

Unknown

Western blot - Anti-Nectin 2 antibody [EPR6717] (AB135246)

The molecular weight observed is consistent with what has been described in the literature (PMID : 15108322, PMID : 12445825 and PMID : 12913096).

Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Nectin 2 antibody [EPR6717] (ab135246) at 1/1000 dilution

Lane 1:

HT-29 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

Mouse brain lysate at 20 µg

Lane 3:

Rat brain 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: 58 kDa

Observed band size: 70-85 kDa

false

• WB

Lab

Western blot - Anti-Nectin 2 antibody [EPR6717] (AB135246)

Western blot : Anti-Nectin 2 antibody [EPR6717] ab135246 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH ab8245 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 64-75 kDa in Wild-type A549 UNBOILED cell lysates with no signal observed at this size in NECTIN2 knockout A549 UNBOILED cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Nectin 2 antibody [EPR6717] (ab135246) at 1/1000 dilution

Lane 1:

Wild-type A549 UNBOILED at 20 µg

Lane 2:

Western blot - Human NECTIN2 knockout A549 cell line (<a href='/en-us/products/cell-lines/human-nectin2-knockout-a549-cell-line-ab288906'>ab288906</a>) at 20 µg

Lane 2:

NECTIN2 knockout A549 UNBOILED cell line

Lane 3:

MCF7 UNBOILED at 20 µg

Lane 4:

Raji UNBOILED at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 58 kDa

Observed band size: 64-75 kDa,37 kDa

false

• WB

Unknown

Western blot - Anti-Nectin 2 antibody [EPR6717] (AB135246)

The molecular weight observed is consistent with what has been described in the literature (PMID : 15108322, PMID : 12445825 and PMID : 12913096).

Blocking Buffer and concentration : 5% NFDM/TBST

All lanes:

Western blot - Anti-Nectin 2 antibody [EPR6717] (ab135246) at 1/1000 dilution

All lanes:

SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysate at 15 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: 58 kDa

Observed band size: 70-85 kDa

false