702283

antibody from Invitrogen Antibodies

Targeting: TBK1 NAK

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Antibody Data

Product number

702283

Provider

Invitrogen Antibodies

Product name

TBK1 Recombinant Rabbit Monoclonal Antibody (10H15L13)

Antibody type

Monoclonal

Antigen

Synthetic peptide

Reactivity

Human, Mouse, Rat

Host

Rabbit

Isotype

IgG

Antibody clone number

10H15L13

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 was performed using Anti-TBK1 Recombinant Rabbit Monoclonal Antibody (10H15L13) (Product # 702283) and a 83 kDa band corresponding to TBK1 was observed across cell lines and tissues tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), K-562 (Lane 2), MCF7 (Lane 3), T-47D (Lane 4) and SK-BR-3 (Lane 5), tissue extracts of Mouse Heart (Lane 7), Rat Heart (Lane 6), Mouse Testis (Lane 8) and Rat Testis (Lane 9) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

For immunofluorescence analysis, SH-SY5Y cells were fixed and permeabilized for detection of endogenous TBK1/NAK using TBK1 NAK Recombinant Rabbit Monoclonal Antibody (Product # 702283, 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 TBK1/NAK 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 TBK1/NAK. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of TBK1 was performed using 70% confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with TBK1 Recombinant Rabbit Monoclonal Antibody (10H15L13) (Product # 702283) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with Hoechst 33342 (Product # H1399). F-actin (Panel c: Red) was stained with Alexa Fluor™ Plus 647 Phalloidin (Product # A30107, 1:2000 dilution). Panel d represents the merged image showing cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 40X magnification in CellInsight CX7 LZR High-Content Screening (HCS) Platform (Product # CX7A1110LZR) and externally deconvoluted (D.Sage et al. / Methods 115 (2017) 28–41).