Anti-Lck antibody [EPR20798-107] - BSA and Azide free

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

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

• IP

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Immunoprecipitation - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

All lanes:

Immunoprecipitation - Anti-Lck antibody [EPR20798-107] (<a href='/en-us/products/primary-antibodies/lck-antibody-epr20798-107-ab227975'>ab227975</a>)

Predicted band size: 58 kDa

Observed band size: 58 kDa

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• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

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] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

• WB

Lab

Western blot - Anti-Lck antibody [EPR20798-107] - BSA and Azide free (AB229379)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab227975).

All lanes:

Western blot - Anti-Lck antibody [EPR20798-107] (<a href='/en-us/products/primary-antibodies/lck-antibody-epr20798-107-ab227975'>ab227975</a>) 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

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