Anti-Lck antibody [EPR20798-107]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Lck antibody [EPR20798-107] (AB227975)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized Ramos (human Burkitt's lymphoma cell line) cells labeling Lck with ab227975 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining on Ramos cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [EPR20798-107] (AB227975)

Immunohistochemical analysis of paraffin-embedded human diffuse large B-cell lymphoma tissue labeling Lck with ab227975 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), ready to use. Membranous and cytoplasmic staining in human diffuse large B-cell lymphoma is observed (PMID : 16769579). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP), ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Lck antibody [EPR20798-107] (AB227975)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized Jurkat (human T cell leukemia cell line from peripheral blood) cell line labeling Lck with ab227975 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

Unknown

Immunoprecipitation - Anti-Lck antibody [EPR20798-107] (AB227975)

Lck was immunoprecipitated from 0.35 mg of Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate with ab227975 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab227975 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1 : Jurkat whole cell lysate 10 μg (Input).
Lane 2 : ab227975 IP in Jurkat whole cell lysate.
Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab227975 in Jurkat whole cell lysate.

Exposure time : 8 seconds.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

All lanes:

Immunoprecipitation - Anti-Lck antibody [EPR20798-107] (ab227975)

Predicted band size: 58 kDa

Observed band size: 58 kDa

true

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [EPR20798-107] (AB227975)

Immunohistochemical analysis of paraffin-embedded rat colon tissue labeling Lck with ab227975 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), ready to use. Membranous and cytoplasmic staining in T cells of rat colon is observed (PMID : 16769579). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP), ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Lck antibody [EPR20798-107] (AB227975)

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized WEHI-231 (mouse lymphoblast B cell lymphoma cell line) cell line labeling Lck with ab227975 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [EPR20798-107] (AB227975)

Immunohistochemical analysis of paraffin-embedded mouse spleen tissue labeling Lck with ab227975 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP), ready to use. Membranous and cytoplasmic staining in mouse spleen reactive lymph node and T cells is observed (PMID : 16769579). Counter stained with Hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP), ready to use.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Lck antibody [EPR20798-107] (AB227975)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized WEHI-231 (mouse lymphoblast B cell lymphoma cell line) cells labeling Lck with ab227975 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining on WEHI-231 cell line.

The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution (red).

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• WB

Unknown

Western blot - Anti-Lck antibody [EPR20798-107] (AB227975)

Exposure times : Lanes 1,2,7 : 5 seconds; Lanes 3,4,8 : 1 second; Lane 5 : 3 seconds; Lane 6 : 10 seconds.

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Lck antibody [EPR20798-107] (ab227975) at 1/1000 dilution

Lane 1:

Human thymus tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Lane 3:

WEHI-231 (mouse lymphoblast B cell lymphoma cell line) whole cell lysate at 20 µg

Lane 4:

Rat thymus tissue lysate at 20 µg

Lane 5:

Mouse lymph node tissue lysate at 20 µg

Lane 6:

Raji (human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 7:

Ramos (human Burkitt's lymphoma cell line) whole cell lysate at 20 µg

Lane 8:

Jurkat (human T cell leukemia cell line from peripheral blood) 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 1/100000 dilution

Predicted band size: 58 kDa

Observed band size: 58 kDa

true

• WB

Supplier Data

Western blot - Anti-Lck antibody [EPR20798-107] (AB227975)

The expression of Lck (phospho Y394) is downregulated in response to Dasatinib treatment.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Lck antibody - Total protein control (ab227975) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Lck (phospho Y394) antibody [EPR27974-93] (<a href='/en-us/products/primary-antibodies/lck-phospho-y394-antibody-epr27974-93-ab318960'>ab318960</a>) at 1/1000 dilution

Lane 1:

COLO 201 (human colon adenocarcinoma tumor ascitic fibroblast-like cell) treated with 200 nM Dasatinib for 24 hours whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Untreated COLO 201 whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated COLO 201 whole cell lysate (alkaline phosphatase treated membrane) 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 1/100000 dilution

Observed band size: 60 kDa,36 kDa

false

Exposure time: 59s

• WB

Lab

Western blot - Anti-Lck antibody [EPR20798-107] (AB227975)

False colour image of Western blot : Anti-Lck antibody [EPR20798-107] 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, ab227975 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 [EPR20798-107] (ab227975) at 1/1000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 20 µg

Lane 2:

Lck knockout Jurkat cell lysate at 20 µg

Lane 2:

Western blot - Human LCK knockout Jurkat cell line (<a href='/en-us/products/cell-lines/human-lck-knockout-jurkat-cell-line-ab273855'>ab273855</a>)

Lane 3:

Ramos cell lysate at 20 µg

Lane 4:

A549 cell lysate at 20 µg

Predicted band size: 58 kDa

Observed band size: 60 kDa

false

• WB

Supplier Data

Western blot - Anti-Lck antibody [EPR20798-107] (AB227975)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Lck antibody - Total protein control (ab227975) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Lck (phospho Y394) antibody [EPR27974-93] (<a href='/en-us/products/primary-antibodies/lck-phospho-y394-antibody-epr27974-93-ab318960'>ab318960</a>) at 1/1000 dilution

Lane 1:

COLO 205 (human colon adenocarcinoma tumor ascitic epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

COLO 205 whole cell lysate (alkaline phosphatase treated membrane) 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 1/100000 dilution

Observed band size: 60 kDa,36 kDa

false

Exposure time: 180s