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Western blot
ELISAAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
- Flow cytometry [1]
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- Product number
- MA5-28676 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- TOLLIP Monoclonal Antibody (SB40a)
- Antibody type
- Monoclonal
- Antigen
- Recombinant full-length protein
- Reactivity
- Human, Mouse
- Host
- Mouse
- Isotype
- IgG
- Antibody clone number
- SB40a
- Vial size
- 200 µL
- Concentration
- 0.5 mg/mL
- Storage
- 4° C
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot was performed using Anti-TOLLIP Monoclonal Antibody (SB40a) (Product # MA5-28676) 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 (1:1000) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).
- 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 Monoclonal Antibody (SB40a) (Product # MA5-28676, 1:2000 ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 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
- 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 10 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with TOLLIP Monoclonal Antibody (SB40a) (Product # MA5-28676) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32766), (1:2000), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). 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.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Flow cytometry analysis of TOLLIP in human T cell leukemia cell line Jurkat. Cells were stained using TOLLIP Monoclonal Antibody (SB40a) (Product # MA5-28676) followed by Goat Anti-Mouse IgG2a, Human ads-PE.
