Anti-Lck antibody [Y123]

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Lck antibody [Y123] (AB32149)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling Lck with purified ab32149 at 1 : 500 dilution (0.8μg/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). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [Y123] (AB32149)

Unpurified ab32149 at a 1/250 dilution staining Lck in human lymphoma using Immunohistochemistry Paraffin Embedded Tissue.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Lck antibody [Y123] (AB32149)

Overlay histogram showing Jurkat cells stained with unpurified ab32149 (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 (ab32149 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5000 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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Lck antibody [Y123] (AB32149)

Intracellular Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling Lck with purified ab32149 at 1/40 dilution (10 ug/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) secondary antibody was used at 1/2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [Y123] (AB32149)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human Hodgkin lymphoma tissue sections labeling Lck with Purified ab32149 at 1 : 250 dilution (1.5 μg/ml). Heat mediated antigen retrieval was performed using heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer pH 9.0). Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• IP

Lab

Immunoprecipitation - Anti-Lck antibody [Y123] (AB32149)

ab32149 (purified) at 1 : 20 dilution (2ug) immunoprecipitating Lck in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate.
Lane 1 (input) : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10ug
Lane 2 (+) : ab32149 & Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab32149 in Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1 : 1000 dilution.
Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Lck antibody [Y123] (ab32149)

Predicted band size: 58 kDa

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• WB

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Western blot - Anti-Lck antibody [Y123] (AB32149)

All lanes:

Western blot - Anti-Lck antibody [Y123] (ab32149) at 1/1000 dilution

All lanes:

Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate

Predicted band size: 58 kDa

Observed band size: 58 kDa

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• WB

Lab

Western blot - Anti-Lck antibody [Y123] (AB32149)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Lck antibody [Y123] (ab32149) at 1/1000 dilution

All lanes:

BxPC-3 (Human pancreas adenocarcinoma epithelial cell) whole cell lysates 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

false

• WB

Lab

Western blot - Anti-Lck antibody [Y123] (AB32149)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Lck antibody [Y123] (ab32149) at 1/5000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 20 µg

Lane 2:

HuT-78 (Human Sezary syndrome cutaneous T lymphocyte) whole cell lysates at 20 µg

Lane 3:

Ramos (Human Burkitt's lymphoma B lymphocyte) whole cell lysates 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

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• WB

Lab

Western blot - Anti-Lck antibody [Y123] (AB32149)

False colour image of Western blot : Anti-Lck antibody [Y123] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32149 was shown to bind specifically to Lck. A band was observed at 60 kDa in wild-type Jurkat cell lysates with no signal observed at this size in Lck knockout cell line ab273855 (knockout cell lysate ab273809). To generate this image, wild-type and Lck knockout Jurkat 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-Lck antibody [Y123] (ab32149) at 1/1000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 20 µg

Lane 2:

Western blot - Human LCK knockout Jurkat cell lysate (<a href='/en-us/products/cell-lysates/human-lck-knockout-jurkat-cell-lysate-ab273809'>ab273809</a>) at 20 µg

Lane 3:

Ramos cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/20000 dilution

Lanes 1 - 4:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 58 kDa

Observed band size: 60 kDa,37 kDa

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