PA5-28697

antibody from Invitrogen Antibodies

Targeting: ZAP70 SRK, STD, ZAP-70

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-28697

Provider

Invitrogen Antibodies

Product name

Zap-70 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Recombinant protein fragment

Description

Recommended positive controls: Jurkat.

Concentration

1.21 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of ZAP70 using 30 µg Jurkat whole cell lysate. Samples were loaded onto a 7.5% SDS-PAGE gel and probed with a ZAP70 polyclonal antibody (Product # PA5-28697) at a dilution of 1:3000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Zap-70 Polyclonal Antibody(Product # PA5-28697) and a 70kDa band corresponding to Zap-70 was observed across cell lines and tissues tested. Membrane enriched extracts (30 µg lysate) of Jurkat (Lane 1), MOLT-4 (Lane 2), Raji (Lane 3), EL4 (Lane 4) and MEF (Lane 5). Tissue Extracts of Mouse Thymus (Lane 6), Rat Thymus (Lane 7), Mouse Liver (Lane 8) and Rat Liver (Lane 19) 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-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunocytochemistry-Immunofluorescence analysis of Zap-70 was performed in Jurkat cells fixed in 4% paraformaldehyde at RT for 15 min. Green: Zap-70 Polyclonal Antibody (Product # PA5-28697) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Zap-70 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 15 minutes at room temperature. The cells were labeled with Zap-70 Polyclonal Antibody (Product # PA5-28697) 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 488 (Product # A32790), (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 with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing membrane localization. Panel e represents Raji cells with no expression of Zap-70. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of paraffin-embedded U373 xenograft, using ZAP70 (Product # PA5-28697) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunoprecipitation of ZAP70 was performed in Jurkat whole cell extracts using 5 µg of Zap-70 Polyclonal Antibody (Product # PA5-28697). Samples were transferred to a membrane and probed with Zap-70 Polyclonal Antibody as a primary antibody and an HRP-conjugated anti-Rabbit IgG was used as a secondary antibody.