702477

antibody from Invitrogen Antibodies

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

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

702477

Provider

Invitrogen Antibodies

Product name

14-3-3 zeta Recombinant Rabbit Monoclonal Antibody (1H9L19)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Antibody clone number

1H9L19

Vial size

100 µg

Concentration

0.5 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 analysis was performed on Tissue extracts (30 µg lysate) of Mouse Brain (Lane 1), Rat Brain (Lane 2) and whole cell extracts (30 µg lysate) of SHSY-5Y (Lane 3), MCF-7 (Lane 4), A-431 (Lane 5), A549 (Lane 6), HeLa (Lane 7) and COS-7 (Lane 8). The blots were probed with Anti-14-3-3 delta/zeta Recombinant Rabbit Monoclonal Antibody (Product # 702477, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 28 kDa band corresponding to 14-3-3 delta/zeta was observed across the tissues and cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, T98G cells were fixed and permeabilized for detection of endogenous 14-3-3 delta/zeta using Anti-14-3-3 delta/zeta Recombinant Rabbit Monoclonal Antibody (Product # 702477, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of 14-3-3 delta/zeta protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of 14-3-3 delta/zeta. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.