Anti-N WASP antibody [EPR6959]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-N WASP antibody [EPR6959] (AB126626)

Intracellular Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labeling N WASP with purified ab126626 at 1/150 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat Anti-Rabbit IgG H&L (Alexa Fluorr^® 488) (ab150077)was used as the secondary antibody. Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-N WASP antibody [EPR6959] (AB126626)

Unpurified ab126626, at 1/50, staining N WASP in formalin fixed, paraffin embedded human papillary carcinoma tissue by Immunohistochemistry

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-N WASP antibody [EPR6959] (AB126626)

Immunofluorescence staining of K562 cells with purified ab126626 at a working dilution of 1/300, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab126626 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-N WASP antibody [EPR6959] (AB126626)

Immunohistochemical staining of paraffin embedded human kidney with purified ab126626 at a working dilution of 1/100. The secondary antibody used is ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

• WB

Lab

Western blot - Anti-N WASP antibody [EPR6959] (AB126626)

Blocking buffer : 5% NFDM/TBST
Dilution buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-N WASP antibody [EPR6959] (ab126626) at 1/10000 dilution

Lane 1:

HeLa whole cell lysate at 10 µg

Lane 2:

K562 whole cell lysate at 10 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: 35 kDa,38 kDa,42 kDa,50 kDa,55 kDa

Observed band size: 42 kDa,65 kDa

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• WB

Lab

Western blot - Anti-N WASP antibody [EPR6959] (AB126626)

Lane 1 : Wild-type HAP1 cell lysate (20 μg)
Lane 2 : N WASP knockout HAP1 cell lysate (20 μg)
Lane 3 : A549 cell lysate (20 μg)
Lane 4 : Human thyroid tissue lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab126626 observed at 67 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab126626 was shown to recognize N WASP when N WASP knockout samples were used, along with additional cross-reactive bands. Wild-type and N WASP knockout samples were subjected to SDS-PAGE. ab126626 and ab8245 (loading control to GAPDH) were diluted 1/1000 and 1/2000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

All lanes:

Western blot - Anti-N WASP antibody [EPR6959] (ab126626)

Predicted band size: 55 kDa

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• WB

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Western blot - Anti-N WASP antibody [EPR6959] (AB126626)

All lanes:

Western blot - Anti-N WASP antibody [EPR6959] (ab126626) at 1/1000 dilution

Lane 1:

NIH 3T3 cell lysate at 10 µg

Lane 2:

F9 cell lysate at 10 µg

Lane 3:

L929 cell lysate at 10 µg

Lane 4:

HeLa cell lysate at 10 µg

Lane 5:

K562 cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP conjugated antibody at 1/2000 dilution

Predicted band size: 55 kDa

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• WB

Lab

Western blot - Anti-N WASP antibody [EPR6959] (AB126626)

Western blot : Rabbit Monoclonal[EPR6959] to N WASP ab126626 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 62 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in WASL knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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-N WASP antibody [EPR6959] (ab126626) at 1/1000 dilution

Lane 1:

Wild-type U-87 MG at 20 µg

Lane 2:

WASL knockout U-87 MG at 20 µg

Secondary

All lanes:

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

Predicted band size: 55 kDa

Observed band size: 62 kDa,36 kDa

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• WB

Lab

Western blot - Anti-N WASP antibody [EPR6959] (AB126626)

All lanes:

Western blot - Anti-N WASP antibody [EPR6959] (ab126626) at 1/10000 dilution

All lanes:

L929 whole cell lysate at 10 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: 55 kDa

Observed band size: 65 kDa

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• OI-RD Scanning

Unknown

OI-RD Scanning - Anti-N WASP antibody [EPR6959] (AB126626)

We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody. Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.