PA5-30747

antibody from Invitrogen Antibodies

Targeting: TOLLIP IL-1RAcPIP

Western blot (Supportive data in Antibodypedia)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Data presented on provider website)

Antibody Data

Product number

PA5-30747

Provider

Invitrogen Antibodies

Product name

TOLLIP Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Recombinant protein fragment

Description

Recommended positive controls: NT2D1, IMR32, U87-MG, MCF-7, CL1-5. Predicted reactivity: Mouse (90%), Rat (90%), Pig (88%), Chicken (87%), Rhesus Monkey (100%), Bovine (89%). Store product as a concentrated solution. Centrifuge briefly prior to opening the vial.

Reactivity

Human, Mouse

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

0.63 mg/mL

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

References

Submitted references

TOLLIP deficiency is associated with increased resistance to Legionella pneumophila pneumonia.

Shah JA, Emery R, Lee B, Venkatasubramanian S, Simmons JD, Brown M, Hung CF, Prins JM, Verbon A, Hawn TR, Skerrett SJ
Mucosal immunology 2019 Nov;12(6):1382-1390

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot using TOLLIP Polyclonal Antibody (Product # PA5-30747). Sample (30 µg of whole cell lysate). Lane A: NT2D1. Lane B: IMR32. 12% SDS PAGE. TOLLIP Polyclonal Antibody (Product # PA5-30747) diluted at 1:1,000.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Knockdown of Toll-interacting protein was achieved by transfecting U-87 MG with Toll-interacting protein specific siRNAs (Silencer® select Product # S29037, S29038). Western Blot analysis (Fig. a) was performed using Whole cell extracts from the Toll-interacting protein knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with TOLLIP Polyclonal Antibody (Product # PA5-30747, 1:3000 ) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:8000). Densitometric analysis of this Western Blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Toll-interacting protein.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western Blot was performed using Anti-TOLLIP Polyclonal Antibody (Product # PA5-30747) and a 32 kDa band corresponding to Toll-interacting protein was observed across the three cell lines and one tissue model. Whole cell extracts (30 µg lysate) of LNCaP (Lane 1), U-87 MG (Lane 2), HeLa (Lane 3), Mouse Liver (Lane 4), Mouse Brain (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:1500) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of Toll-interacting protein was performed using 70% confluent log phase U-87 MG cells. The cells were fixed with 4% paraformaldehyde for 15 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 TOLLIP Polyclonal Antibody (Product # PA5-30747) at 1:100 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), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Cytoplasm localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60x magnification.