711208

antibody from Invitrogen Antibodies

Targeting: KRT8 CARD2, CK8, CYK8, K2C8, K8, KO

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

711208

Provider

Invitrogen Antibodies

Product name

Phospho-Cytokeratin 8 (Ser432) Recombinant Polyclonal Antibody (21HCLC)

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Monkey and squirrel.

Antibody clone number

21HCLC

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 Caco-2 (Lane 1), Caco-2 treated with EGF (100 ng/mL for 20mins) (Lane 2), A549 (Lane 3) and A549 treated with EGF (100 ng/mL for 20mins) (Lane 4). The blots were probed with Anti-Cytokeratins 8 (pS342) Recombinant Rabbit Polyclonal Antibody (Product # 711208, 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 59 kDa band corresponding to Cytokeratins 8 (pS342) 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, Caco-2 cells were fixed and permeabilized for detection of endogenous Cytokeratin 8 (pS342) using Cytokeratins 8 (pS342) Recombinant Rabbit Polyclonal Antibody (Product # 711208, 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 Cytokeratin 8 (pS342) (green) in the cytoplasm and cytoskeleton can be clearly observed in cells treated with EGF (100 ng/mL, 20 min), as compared to untreated cells. Antibody specificity was demonstrated by competition with the Cytokeratin 8 (pS342) phosphopeptide, which results in loss of signal. No competition was observed with the non-phospho peptide. The images were captured at 60X magnification.