- MA5-15814 - Invitrogen Antibodies RUNX1 antibody

Palangat M, Anastasakis DG, Fei DL, Lindblad KE, Bradley R, Hourigan CS, Hafner M, Larson DR
Genes & development 2019 May 1;33(9-10):482-497

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockdown of RUNX1 was achieved by transfecting Jurkat with RUNX1 specific siRNAs (Silencer® select Product # s229351, s2458). Western blot analysis (Fig. a) was performed using modified whole cell extracts (1% SDS) from the RUNX1 knockdown cells (lane 3), non-specific scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with RUNX1 Monoclonal Antibody (5A1) (Product # MA5-15814, 1:1000 dilution) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP conjugate (Product # A28177, 0.25 µg/mL, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to RUNX1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot was performed using Anti-RUNX1 Monoclonal Antibody (5A1), (Product # MA5-15814) and a 55 kDa band corresponding to RUNX1 along with uncharacterized bands (*) at ~70 kDa and ~200 kDa were observed in the cell lines tested. Modified whole cell extracts (1% SDS) (40 µg lysate) of Jurkat (Lane 1), HEL 92.1.7 (Lane 2), T-47D (Lane 3) and IMR32 (Lane 4) were electrophoresed using Novex® NuPAGE® 4-12 % Bis-Tris gel (Product # NP0321BOX). 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).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis of RUNX1 using RUNX1 monoclonal antibody (Product # MA5-15814) in Jurkat cell lysate.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of RUNX1 was performed using 70% confluent log phase Jurkat 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 1 hour at room temperature. The cells were labeled with RUNX1 Monoclonal Antibody (5A1) (Product # MA5-15814) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766) at a dilution of 1:2000 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). Panel d represents the merged image showing predominantly nuclear localization. Panel e 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: Immunofluorescence analysis of HeLa cells using RUNX1 monoclonal antibody (Product # MA5-15814) (Green). Red: actin filaments have been labeled with phalloidin.

MA5-15814