PA1-4639

antibody from Invitrogen Antibodies

Targeting: RAF1 c-Raf, CRAF, Raf-1

Western blot (Supportive data in Antibodypedia)

Antibody Data

Product number

PA1-4639

Provider

Invitrogen Antibodies

Product name

Phospho-c-Raf (Ser301) Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with bovine, canine, chicken, mouse, non-human primate and Xenopus based on 100% sequence homology.

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot of UV treated human Jurkat cell lysate showing specific immunolabeling of the ~74 kDa Raf-1 protein (Lane 1). The phosphospecificty of this labeling is shown in Lane 2 (lambda-phosphatase). The blot is identical to the control except that it was incubated in lambda-Ptase (1200 units for 30 min) before being exposed to the Anti-Ser301 Raf-1. The immunolabeling of Raf-1 is completely eliminated by treatment with lambda-Ptase.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis was performed on membrane enriched extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with TPA (200 nM for 30 minutes) (Lane 2), COS-7 (Lane 3), COS-7 treated with TPA (200 nM for 30 minutes) (Lane 4), T-47D (Lane 5), T-47D treated with TPA (200 nM for 30 minutes) (Lane 6), NIH/3T3 (Lane 7) and NIH/3T3 treated with TPA (200 nM for 30 minutes) (Lane 8). The blot was probed with Anti-Phospho-RAF1 (Ser301) Rabbit Polyclonal Antibody (Product # PA1-4639, 1:1000 dilution) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/mL, 1:4000 dilution). A 74 kDa band corresponding to Phospho-RAF1 (Ser301) was detected and observed to increase upon TPA treatment across 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 using iBlot® 2 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).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot of c-Raf in UV treated human Jurkat cell lysate showing specific immunolabeling of a band at ~74 kDa corresponding to Phospho-c-Raf (Ser301) polyclonal antibody (Product # PA1-4639) in the first lane (-). Phosphospecificity is shown in the second lane (+) where the immunolabeling is completely eliminated by blot treatment with lambda phosphatase (1,200 units for 30 minutes).