701643

antibody from Invitrogen Antibodies

Targeting: IKBKB IKK-beta, IKK2, IKKB, NFKBIKB

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

701643

Provider

Invitrogen Antibodies

Product name

Phospho-IKK alpha/beta (Ser176, Ser180) Recombinant Rabbit Monoclonal Antibody (7H17L17)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Description

This antibody is predicted to react with Dog, Rabbit, Pig, Mouse, Rat, Bovine and Goat.

Antibody clone number

7H17L17

Concentration

0.5 mg/mL

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence was performed on HeLa cells treated with TNF alpha at 50 ng/mL/20 min. These cells were fixed and permeabilized for detection of IKK alpha/beta (pSpS 176/180) using Anti-IKK alpha/beta (pSpS 176/180) Recombinant Rabbit Monoclonal Antibody (Product # 701643, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of the protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic and nuclear localization of IKK alpha/beta (pSpS 176/180). Panel e) represents control cells with no primary Antibody to assess background. Panel f) shows untreated cells.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Time course showing induction of TNF-a signaling cascade upon treatment: Cellular localization of proteins in the NF-KB signaling pathway was detected upon treatment of HeLa cells with TNF-a (50 ng/mL) for 5, 10 and 25 min, respectively. Fixed and permeabilized cells were stained with Anti-IKK alpha/beta (pSpS176/180) Recombinant Rabbit Monoclonal Antibody (Product # 701643, 1 µg/mL) or Anti-NF-kB Mouse Monoclonal Antibody (Product # 33-9900, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500) and Goat anti-Mouse IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 647 conjugate (Product # A28181, 0.4 µg/mL, 1:2500). Images show staining of phospho-IKK alpha/beta and NF-KB (panel a, e, i, m) in untreated cells. No significant basal levels of phosphorylated IKK alpha/beta (panel a; green) were detected. Treatment with TNF alpha led to an increase in the levels of phospho-IKK alpha/beta (panel b - d ; green) in the cytosol and the nucleus, and a corresponding translocation of NF-kB to the nucleus (panel f - h; red). The composite images are shown in panels i - l; green, red. Nuclei (blue) were stained using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50) and panel m - p; green, red represent the specific localization of the proteins with reference to DAPI. No background staining was observed in control cells with no primary antibody (panel q).

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Phospho-IKK alpha/beta (Ser176, Ser180) was done on 70% confluent log phase ME-180 cells. The cells were either mock treated or treated with TNF-alpha (50 ng/mL for 20 min), fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 1% BSA for 1 hour at room temperature. The cells were subsequently labeled with Phospho-IKK alpha/beta (Ser176, Ser180 - Green) Rabbit Monoclonal Antibody (Product # 701643) at 2µg/mL in 0.1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature. Nuclei (Blue) were stained with SlowFade® Gold Antifade Mountant DAPI (Product # S36938). F-actin (Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). TNFalpha induced phosphorylation of IKK alpha/beta, was observed in control cell line (panels a, c) and not in the IKK alpha knockout (KO) cell line (panels b, d). The images were captured at 40X magnification.