- MA5-12303 - Invitrogen Antibodies LCK antibody

Submitted references Robust fluorescent detection of protein fatty-acylation with chemical reporters.

Charron G, Zhang MM, Yount JS, Wilson J, Raghavan AS, Shamir E, Hang HC
Journal of the American Chemical Society 2009 Apr 8;131(13):4967-75

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot of LCK (P56lck) using LCK (P56lck) Monoclonal Antibody (Product # MA5-12303) on Jurkat Cells.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-LCK Monoclonal Antibody (3A5) (Product # MA5-12303) and a 55 kDa band corresponding to LCK was observed across was observed in all cells and tissues tested except A549, THP-1, Mouse Brain, Rat Brain and Mouse testis which are reported to be low to negative for LCK expression. An additional uncharacterized band (*) around 110 kDa was observed in Brain tissues, while IgG was observed in Mouse Testis and Mouse Thymus. Membrane enriched extracts (30 µg lysate) of Jurkat (Lane 1), MOLT-4 (Lane 2), A549 (Lane 3), THP-1 (Lane 4), Mouse Brain (Lane 5), Rat Brain (Lane 6), Mouse Testis (Lane 7), Mouse Thymus (Lane 8) and Rat Thymus (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1ug/ml) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177,1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of LCK (p56lck) (green) showing staining in the cytoplasm of BaF-3 cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LCK (p56lck) monoclonal antibody (Product # MA5-12303) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of LCK (p56lck) (green) showing staining in the cytoplasm of Jurkat cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LCK (p56lck) monoclonal antibody (Product # MA5-12303) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescent analysis of LCK (p56lck) (green) showing staining in the cytoplasm of Ramos cells (right) compared to a negative control without primary antibody (left). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5-10 minutes and blocked with 3% BSA-PBS for 30 minutes at room temperature. Cells were probed with a LCK (p56lck) monoclonal antibody (Product # MA5-12303) in 3% BSA-PBS at a dilution of 1:20 and incubated overnight at 4ºC in a humidified chamber. Cells were washed with PBST and incubated with a DyLight-conjugated secondary antibody in PBS at room temperature in the dark. Nuclei were stained with Hoechst or DAPI (blue). Images were taken at a magnification of 60x.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of LCK was performed using 70% confluent log phase Jurkat and THP-1 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with LCK Monoclonal Antibody (3A5) (Product # MA5-12303) at 1:100 in 0.1% BSA, incubated at 4-degree Celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Plasma membrane localization. Panel e represents THP-1 cells which are reported to be low to negative for LCK expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of LCK (p56lck) in BAF-3 cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a LCK (p56lck) monoclonal antibody (Product # MA5-12303) at a dilution of 1 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of LCK (p56lck) in Jurkat cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a LCK (p56lck) monoclonal antibody (Product # MA5-12303) at a dilution of 1 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Flow cytometry analysis of LCK (p56lck) in Ramos cells (green) compared to an isotype control (blue). Cells were harvested, adjusted to a concentration of 1-5x10^6 cells/mL, fixed with 2% paraformaldehyde and washed with PBS. Cells were blocked with a 2% solution of BSA-PBS for 30 min at room temperature and incubated with a LCK (p56lck) monoclonal antibody (Product # MA5-12303) at a dilution of 1 µg/test for 40 min at room temperature. Cells were then incubated for 40 min at room temperature in the dark using a Dylight 488-conjugated secondary antibody and re-suspended in PBS for FACS analysis.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunoprecipitation of LCK (P56lck) using LCK (P56lck) Monoclonal Antibody (Product # MA5-12303) on denatured Human Lck Cells.

MA5-12303

Antibody data