重组Anti-Nck抗体[Y531]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Nck antibody [Y531] (AB32120)

Overlay histogram showing Jurkat cells stained with ab32120 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab32120, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Nck antibody [Y531] (AB32120)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (human cervix adenocarcinoma epithelial cell) cells labeling Nck with purified ab32120 at 1/250 dilution (8 μg/mL). Cells were fixed in 100% Methanol. 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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Nck antibody [Y531] (AB32120)

ab32120 (1/100), staining human breast carcinoma by immunohistochemistry, Paraffin embedded tissue.

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

• WB

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Western blot - Anti-Nck antibody [Y531] (AB32120)

All lanes:

Western blot - Anti-Nck antibody [Y531] (ab32120) at 1/10000 dilution

All lanes:

Hela cell lysate

Predicted band size: 43 kDa

Observed band size: 35 kDa,47 kDa

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• WB

Lab

Western blot - Anti-Nck antibody [Y531] (AB32120)

Lanes 1-3 : Merged signal (red and green). Green - ab32120 observed at 50 kDa. Red - loading control ab8245 observed at 36 kDa.

ab32120 Anti-Nck antibody [Y531] was shown to specifically react with Nck in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265323 (knockout cell lysate ab258059) was used. Wild-type and Nck knockout samples were subjected to SDS-PAGE. ab32120 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 1 in 20000 dilution respectively. 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Nck antibody [Y531] (ab32120) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

NCK1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human NCK1 (Nck) knockout HeLa cell line (<a href='/products/cell-lines/human-nck1-nck-knockout-hela-cell-line-ab265323'>ab265323</a>)

Lane 3:

NIH/3T3 cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 43 kDa

Observed band size: 50 kDa

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