MA1-25665

antibody from Invitrogen Antibodies

Targeting: YWHAZ 14-3-3-zeta, KCIP-1, YWHAD

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Recommended by provider)

Antibody Data

Product number

MA1-25665

Provider

Invitrogen Antibodies

Product name

14-3-3 beta/zeta Monoclonal Antibody (4E2)

Antibody type

Monoclonal

Antigen

Other

Description

Recommended positive controls: HeLa cell lysate.

Reactivity

Human, Mouse, Rat, Bovine, Canine, Porcine

Host

Mouse

Isotype

IgG

Antibody clone number

4.00E+02

Vial size

50 µg

Concentration

1 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

Western blot was performed using Anti-14-3-3 beta/zeta Monoclonal Antibody (4E2) (Product # MA1-25665) and a 28 kDa band corresponding to 14-3-3 protein beta/zeta was observed across the cell lines and tissues tested. Membrane enriched extracts (30 µg lysate) of MCF7 (Lane 1), HeLa (Lane 2), PC-3 (Lane 3), A549 (Lane 4), Mouse Skeletal Muscle (Lane 5), Rat Skeletal Muscle (Lane 6), Mouse Brain (Lane 7), Rat Brain (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10,000 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

Knockdown of 14-3-3 protein beta/zeta was achieved by transfecting MCF7 with 14-3-3 protein beta/zeta specific siRNAs (Silencer® select Product # s14961, s14962, s14972). Western blot analysis (Fig. a) was performed using Membrane enriched extracts from the 14-3-3 protein beta/zeta knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with 14-3-3 beta/zeta Monoclonal Antibody (4E2) (Product # MA1-25665, 1 µg/mL ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10,000 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 14-3-3 protein beta/zeta.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-14-3-3 beta/zeta Monoclonal Antibody (4E2) (Product # MA1-25665) and a 28 kDa band corresponding to 14-3-3 protein beta/zeta was observed across the cell lines and tissues tested. Membrane enriched extracts (30 µg lysate) of MCF7 (Lane 1), HeLa (Lane 2), PC-3 (Lane 3), A549 (Lane 4), Mouse Skeletal Muscle (Lane 5), Rat Skeletal Muscle (Lane 6), Mouse Brain (Lane 7), Rat Brain (Lane 8) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 µg/mL) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:10,000 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

Immunofluorescence analysis of 14-3-3 protein beta/zeta was performed using 70% confluent log phase MCF7 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 14-3-3 beta/zeta Monoclonal Antibody (4E2) (Product # MA1-25665) at 1:50 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 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 Cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.