700727

antibody from Invitrogen Antibodies

Targeting: GATA1 ERYF1, GATA-1, GF1, NF-E1, NFE1

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

700727

Provider

Invitrogen Antibodies

Product name

GATA1 Recombinant Rabbit Monoclonal Antibody (4H15L5)

Antibody type

Monoclonal

Antigen

Recombinant full-length protein

Description

Intact IgG appears on a non-reducing gel as ~150 kDa band and upon reduction generating a ~25 kDa light chain band and a ~50 kDa heavy chain.

Antibody clone number

4H15L5

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-GATA1 Recombinant Rabbit Monoclonal Antibody (4H15L5) (Product # 700727) and a 50kDa band corresponding to Erythroid transcription factor was observed across in K-562 and HEL 92.1.7 cell lines as opposed to all other models tested. Nuclear enriched extracts (30 µg lysate) of HEL 92.1.7 (Lane 1), K-562 (Lane 2), Jurkat (Lane 3), Raji (Lane 4), A-431 (Lane 5), SK-O-V3 (Lane 6), IMR-32 (Lane 7), Mouse Thymus (Lane 8), Mouse Spleen (Lane 9), HeLa (Lane 10) 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 (2 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20,000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of GATA1 was performed by loading 20 µg of K562 cell lysate using Novex®NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002), Novex® Sharp Pre-Stained Protein Standard (Product # LC5800), and iBlot® Dry Blotting System (Product # IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. GATA1 was detected at ~ 50 kDa using GATA1 Recombinant Rabbit Monoclonal Antibody (Product # 700727) at 1-3 µg/mL in 2.5% skim milk at 4°C overnight on a rocking platform. Goat anti-Rabbit IgG - HRP Secondary Antibody (Product # G-21234) at 1:5000 dilution was used and chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Erythroid transcription factor was performed using log phase K-562 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with GATA1 Recombinant Rabbit Monoclonal Antibody (4H15L5) (Product # 700727) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 594 (Product # A32754), (1:2000 dilution), 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 withRhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing nuclear localization. Panel e shows Jurkat cells with no expression of GATA1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification in EVOS™ M7000 Imaging System (Product # AMF7000) and externally deconvoluted (D.Sage et al. / Methods 115 (2017) 28-41).