MA5-32897

antibody from Invitrogen Antibodies

Targeting: AXL ARK, JTK11, Tyro7, UFO

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

MA5-32897

Provider

Invitrogen Antibodies

Product name

Axl Monoclonal Antibody (C4-A8)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Reactivity

Human

Host

Mouse

Isotype

IgG

Antibody clone number

C4-A8

Vial size

100 µL

Concentration

2 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

CRISPR-Cas9 mediated genome editing ofAxl (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR932417_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of AXL was performed by loading 30 µg of U-87 MG Cas9 (Lane 1), and U-87 MG Cas9 cells transduced with Axl Lentiviral sgRNA (Lane 2) membrane enriched extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Axl Monoclonal Antibody (C4-A8) (Product # MA5-32897) using 1:1,000 dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 1:5,000 dilution).Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toAxl (Fig (b)).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Axl in recombinant protein using a Axl Monoclonal antibody (Product # MA5-32897) at a dilution of 1:20,000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Axl Monoclonal Antibody (C4-A8)(Product # MA5-32897) and a 130kDa band corresponding to Axl was observed across cell lines tested. Membrane enriched extracts (30 µg lysate) of U-87 MG (Lane 1), U-2 OS (Lane 2), T98G (Lane 3), SH-SY5Y (Lane 4), HEK-293 (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1000 Dilution) 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).Expression of AXL was found to be higher in U-87 MG,U-2 OS and T98G as compared to SH-SY5Y and HEK-293 cells.

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemical analysis of Axl in SW480 cells using a Axl Monoclonal antibody (Product # MA5-32897).The nuclear counter stain is DAPI (blue). Cells were fixed in paraformaldehyde, permeabilised with 0.25% Triton X100/PBS.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Axl was performed using 70% confluent log phase U-87 MG 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 Axl Monoclonal Antibody (C4-A8) (Product # MA5-32897) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing cell membrane localization. Panel e represents SH-SY5Y with no AXL expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.