702365

antibody from Invitrogen Antibodies

Targeting: CBL c-Cbl, CBL2, RNF55

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

702365

Provider

Invitrogen Antibodies

Product name

Phospho-c-Cbl (Tyr774) Recombinant Rabbit Monoclonal Antibody (8H4L1)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey, Cat and Pig

Antibody clone number

8H4L1

Concentration

0.5 mg/mL

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on whole cell extracts (30 µg lysate) of A549 (Lane 1), A549 treated with Pervanadate (Serum starved for 2 hours and treated for 1 hour with 100 uM Pervanadate) (Lane 2). The blots were probed with Anti-c-Cbl pY774 Recombinant Rabbit Monoclonal Antibody (Product # 702365, 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 ~ 100 kDa band corresponding to c-Cbl pY774 was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by overnight wet transfer. 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, Jurkat cells were fixed and permeabilized for detection of endogenous c-Cbl pY (774) using Anti-c-Cbl pY (774) Recombinant Rabbit Monoclonal Antibody (Product # 702365, 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 c-Cbl pY (774) 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 Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of c-Cbl pY (774). Panel e) shows loss of signal by competition with the c-Cbl pY (774) peptide, demonstrating antibody specificity, and panel f) demonstrates no competition with the non-phospho peptide. Panel g) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.