711717

antibody from Invitrogen Antibodies

Targeting: RAD17 CCYC, RAD17Sp, Rad24

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711717

Provider

Invitrogen Antibodies

Product name

Phospho-RAD17 (Ser656) Recombinant Polyclonal Antibody (12HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Reactivity

Human

Host

Rabbit

Isotype

IgG

Antibody clone number

12HCLC

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 Whole cell extracts (30 µg lysate) of HeLa (Lane 1), HeLa treated with Etoposide (100 µM/mL for 2hr) (Lane 2), K-562 (Lane 3) and K-562 treated with Etoposide (100 µM/mL for 2hr) (Lane 4). The blots were probed with Anti-Rad17 (pS656) Recombinant Rabbit Polyclonal Antibody (Product # 711717, 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 77 kDa band corresponding to Rad17 (pS656) was observed across the 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, HeLa cells were fixed and permeabilized for detection of endogenous RAD17 pS656 using Anti-RAD17 pS656 Recombinant Rabbit Polyclonal Antibody (Product # 711717, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938), and Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300) was used for cytoskeletal F-actin (red) staining. Detection and localization of RAD17 pS656 (green) in the nucleus can be clearly observed in cells treated with etoposide (100 uM, 2 h) as compared to untreated cells. Antibody specificity was demonstrated by competition with the RAD17 pS656 peptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.