Does Human TNF-alpha antibody, clone # 6401, detect membrane bound or soluble TNF-alpha?

Name: Human TNF-alpha Antibody
Brand: R&D Systems
SKU: MAB2101
Price: 459 USD

Human TNF-alpha antibody, Clone # 6401, binds to and neutralizes Catalog # 210-TA, which is an E. coli-derived human TNF-alpha protein, Val77-Leu233, and has been shown to be cytotoxic to L‑929 mouse fibroblast cells. According to Uniprot, the soluble form of TNF-alpha is amino acids 77-233: https://www.uniprot.org/uniprot/P01375. We have also shown binding of Clone # 6401 to the membrane bound form of TNFa via flow cytometry. We are aware of a few citations showing the antibody acts to neutralize TNFa. See https://www.rndsystems.com/products/human-tnf-alpha-antibody-6401_mab2101#product-citations. Therefore, clone #6401 detects both membrane bound and soluble forms of TNF-alpha.

Human TNF-alpha Antibody

Catalog #: MAB2101
13 Citations Datasheet / COA / SDS

Catalog #	Availability	Size / Price	Qty
MAB2101-SP
MAB2101-500
MAB2101-100

Cytotoxicity Induced by TNF‑ alpha and Neutralization by Human TNF‑ alpha Antibody.

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All TNF-alpha Antibodies

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Citations (13)

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Summary Data Examples Preparation and Storage Reconstitution Calculator Background Product Datasheets Related Research Areas

Human TNF-alpha Antibody Summary

Species Reactivity

Human

Specificity

Detects human TNF-alpha in direct ELISAs and Western blots. In direct ELISAs, approximately 25‑50% cross‑reactivity with recombinant porcine TNF‑ alpha and recombinant rhesus macaque TNF-alpha is observed but no cross-reactivity with recombinant cotton rat TNF‑ alpha, recombinant rat TNF‑ alpha, recombinant human (rh) LT alpha 1/ beta 2, rhLT alpha 2/ beta 1, rhAPRIL, rhBAFF, rhEDA‑A2, recombinant mouse EDA, rhFas Ligand, rhLIGHT, rhOX40 Ligand, rhTRAIL, rhTRANCE, rhTWEAK, or rhVEGI is observed.

Source

Monoclonal Mouse IgG₁ Clone # 6401

Purification

Protein A or G purified from ascites

Immunogen

E. coli-derived recombinant human TNF-alpha
Gly57-Leu233 (predicted)
Accession # P01375

Formulation

Lyophilized from a 0.2 μm filtered solution in PBS with Trehalose. *Small pack size (SP) is supplied either lyophilized or as a 0.2 µm filtered solution in PBS.

Endotoxin Level

<0.10 EU per 1 μg of the antibody by the LAL method.

Label

Unconjugated

Applications

Recommended Concentration

Sample

Flow Cytometry

0.25 µg/10⁶ cells

Human PBMC treated with 1 μg/ml LPS for 4 hours

CyTOF-ready

Ready to be labeled using established conjugation methods. No BSA or other carrier proteins that could interfere with conjugation.

Neutralization

Measured by its ability to neutralize TNF‑ alpha -induced cytotoxicity in the L‑929 mouse fibroblast cell line. Matthews, N. and M.L. Neale (1987) in Lymphokines and Interferons, A Practical Approach. Clemens, M.J. et al. (eds): IRL Press. 221. The Neutralization Dose (ND₅₀) is typically 0.015-0.06 µg/mL in the presence of 0.25 ng/mL Recombinant Human TNF‑ alpha and 1 µg/mL actinomycin D.

Please Note: Optimal dilutions should be determined by each laboratory for each application. General Protocols are available in the Technical Information section on our website.

Scientific Data

Neutralization

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Cytotoxicity Induced by TNF‑ alpha and Neutralization by Human TNF‑ alpha Antibody. Recombinant Human TNF-a (Catalog # 210-TA) induces cytotoxicity in the L-929 mouse fibroblast cell line in a dose-dependent manner (orange line). Cytotoxicity elicited by Recombinant Human TNF-a (0.25 ng/mL) is neutralized (green line) by increasing concentrations of Human TNF-a Monoclonal Antibody (Catalog # MAB2101). The ND₅₀ is typically 0.015-0.06 µg/mL in the presence of the metabolic inhibitor actinomycin D (1 µg/mL).

Flow Cytometry

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Detection of TNF‑ alpha in Human PBMC Monocytes by Flow Cytometry. Human PBMCs were treated with 1μg/ml LPS for 4 hours then stained with (A) Mouse Anti-Human TNF-a Membrane Form Monoclonal Antibody (Catalog # MAB2101) or (B) isotype control antibody (MAB002) followed by anti-Mouse IgG PE-conjugated Secondary Antibody (F0102B) and Mouse anti-Human CD14 APC-conjugated Monoclonal Antibody (FAB3832A). Staining was perfomed using our Staining Membrane-associated Proteins protocol.

Western Blot

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Detection of Insect TNF-alpha by Western Blot The pre-integrated TMD docks to the cytosolic tip of the lateral gate.(A) Co-immunoprecipitation of recombinant FLAG-Sec61 alpha and Sec61 gamma. Sf21 insect cell microsomes were solubilized with 1% Deoxy BigChap (DBC) and subjected to anti-FLAG immunoprecipitation (IP). Eluted samples (Sf21) were analyzed by immunoblotting next to a sample of canine microsomal ER membranes (CRM). (B) Comparison of the relative CT7 photo-crosslinking efficiency of canine rough microsomes (CRM) and Sf21 microsomes containing wild-type FLAG-Sec61 alpha / gamma complex. CT7 crosslinking was performed as before. The relative crosslinking efficiency of recombinant Sec61 alpha was normalized to the native protein. (C) BMH crosslinking reactions using recombinant FLAG-Sec61 alpha / gamma complex. Cys49 TNF alpha 126-mers were prepared in the presence of Sf21 microsomes, and BMH crosslinking reactions were conducted as described previously. Reactions were either analyzed directly (Total) or after denaturing IP with anti-FLAG affinity resin. IP with the anti-HA affinity resin served as a specificity control. (D) Western blots of Sf21 microsomes co-expressing either wild-type or mutant FLAG-Sec61 alpha / gamma. Insect cell microsomes from cells infected with a baculovirus that lacked Sec61 genes (control virus) served as a control. (E) CT7 crosslinking of mutant Sec61 alpha / gamma complexes containing the indicated cysteine substitutions. The region of the gel corresponding to FLAG-Sec61 alpha is shown. Detection of photo-affinity labeling that is competed by CT9 indicates that the mutant Sec61 alpha / gamma complex is properly folded.DOI:https://dx.doi.org/10.7554/eLife.01483.010 Image collected and cropped by CiteAb from the following publication (https://elifesciences.org/articles/01483), licensed under a CC-BY license. Not internally tested by R&D Systems.

Reconstitution Calculator

Preparation and Storage

Reconstitution

Reconstitute at 0.5 mg/mL in sterile PBS.

Shipping

Lyophilized product is shipped at ambient temperature. Liquid small pack size (-SP) is shipped with polar packs. Upon receipt, store immediately at the temperature recommended below.

Stability & Storage

Use a manual defrost freezer and avoid repeated freeze-thaw cycles.

12 months from date of receipt, -20 to -70 °C as supplied.
1 month, 2 to 8 °C under sterile conditions after reconstitution.
6 months, -20 to -70 °C under sterile conditions after reconstitution.

Background: TNF-alpha

Tumor necrosis factor alpha (TNF-alpha, TNF- alpha, TNFA ), also known as Cachectin and TNFSF2, is the prototypic ligand of the TNF superfamily. It is a pleiotropic molecule that plays a central role in inflammation, immune system development, apoptosis, and lipid metabolism. TNF- is produced by several lymphoid cells as well as by astrocytes, endothelial cells, and smooth muscle cells. Human TNF-alpha consisits of a 35 amino acid (aa) cytoplasmic domain, a 21 aa transmembrane segment, and a 177 aa extracellular domain (ECD). Within the ECD, human TNF-alpha shares 97% aa sequence identity with rhesus and 71%-92% with bovine, canine, cotton rat, equine, feline, mouse, porcine, and rat TNF-alpha. TNF-alpha is produced by a wide variety of immune, epithelial, endothelial, and tumor cells. TNF-alpha is assembled intracellularly to form a noncovalently linked homotrimer which is expressed on the cell surface. Cell surface TNF-alpha can induce the lysis of neighboring tumor cells and virus infected cells, and it can generate its own downstream cell signaling following ligation by soluble TNFR I. Shedding of membrane bound TNF-alpha by TACE/ADAM17 releases the bioactive cytokine, a 55 kDa molecular weight soluble trimer of the TNF-alpha extracellular domain. TNF-alpha binds the ubiquitous 55-60 kDa TNF RI and the hematopoietic cell-restricted 80 kDa TNF RII, both of which are also expressed as homotrimers present on virtually all cell types. Both type I and type II receptors bind TNF-alpha with comparable affinity, although only TNF RI contains a cytoplasmic death domain which triggers the activation of apoptosis. Soluble forms of both types of receptors are released and can neutralize the biological activity of TNF-alpha.

Long Name

Tumor Necrosis Factor alpha

Entrez Gene IDs

7124 (Human); 21926 (Mouse); 24835 (Rat); 397086 (Porcine); 280943 (Bovine); 403922 (Canine); 102139631 (Cynomolgus Monkey); 100033834 (Equine); 493755 (Feline); 100009088 (Rabbit)

Alternate Names

APC1 protein; Cachectin; Cachetin; DIF; TNF; TNF, monocyte-derived; TNFA; TNF-A; TNFalpha; TNF-alpha; TNF-alphacachectin; TNFATNF, macrophage-derived; TNFG1F; TNFSF1A; TNFSF2; TNFSF2TNF superfamily, member 2; tumor necrosis factor (TNF superfamily, member 2); tumor necrosis factor alpha; Tumor necrosis factor ligand superfamily member 2; tumor necrosis factor; tumor necrosis factor-alpha

Product Datasheets

Product Datasheet

COA

SDS

Related Research Areas

Citations for Human TNF-alpha Antibody

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

13 Citations: Showing 1 - 10
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NFATc2 is an intrinsic regulator of melanoma dedifferentiation.
Authors: V Perotti, P Baldassari, A Molla et al.
Oncogene
Targeting aPKC disables oncogenic signaling by both the EGFR and the proinflammatory cytokine TNFalpha in glioblastoma.
Authors: Kusne Y, Carrera-Silva EA, Perry AS et al.
Sci Signal
Strain-Modulated and Nanorod-Waveguided Fluorescence in Single Zinc Oxide Nanorod-Based Immunodetection
Authors: Sytu, MRC;Stoner, A;Hahm, JI;
Biosensors
Species: N/A
Sample Types: Small Molecule
Applications: ELISA Capture
A bispecific ?? T-cell engager targeting EGFR activates a potent V?9V?2 T cell-mediated immune response against EGFR-expressing tumors
Authors: King, LA;Toffoli, EC;Veth, M;Iglesias-Guimarais, V;Slot, MC;Amsen, D;van de Ven, R;Derks, S;Fransen, MF;Tuynman, JB;Riedl, T;Roovers, RC;Adang, AEP;Ruben, JM;Parren, PWHI;de Gruijl, TD;van der Vliet, HJ;
Cancer immunology research
Species: Human
Sample Types: Whole Cells
Applications: Neutralization
Peficitinib improves bone fragility by recovering bone turnover imbalance in arthritic mice
Authors: S Sugahara, K Hanaoka, T Emori, N Takeshita, Y Fujii, M Nakano, T Suzuki, J Takahashi, Y Nakamura
Journal of pharmacological sciences, 2021-11-03;148(1):134-141.
Species: Human
Sample Types: Whole Cells
Applications: Neutralization
Periodontitis-associated pathogens P. gingivalis and A. actinomycetemcomitans activate human CD14(+) monocytes leading to enhanced Th17/IL-17 responses
Authors: Wan-Chien Cheng
Eur J Immunol, 2016-08-01;0(0):.
Species: Human
Sample Types: Whole Cells
Applications: Neutralization
Vitamin D regulates cytokine patterns secreted by dendritic cells to promote differentiation of IL-22-producing T cells.
Authors: Sommer A, Fabri M
PLoS ONE, 2015-06-24;10(6):e0130395.
Species: Human
Sample Types: Whole Cells
Applications: Neutralization
Innate lymphoid cells integrate stromal and immunological signals to enhance antibody production by splenic marginal zone B cells.
Authors: Magri G, Miyajima M, Bascones S, Mortha A, Puga I, Cassis L, Barra C, Comerma L, Chudnovskiy A, Gentile M, Llige D, Cols M, Serrano S, Arostegui J, Juan M, Yague J, Merad M, Fagarasan S, Cerutti A
Nat Immunol, 2014-02-23;15(4):354-64.
Species: Human
Sample Types: Whole Cells
Applications: Neutralization
Production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory T cells.
Authors: Duhen T, Geiger R, Jarrossay D, Lanzavecchia A, Sallusto F
Nat. Immunol., 2009-07-05;10(8):857-63.
Species: Human
Sample Types: Whole Cells
Applications: Neutralization
Der p 1 suppresses indoleamine 2, 3-dioxygenase in dendritic cells from house dust mite-sensitive patients with asthma.
Authors: Maneechotesuwan K, Wamanuttajinda V, Kasetsinsombat K, Huabprasert S, Yaikwawong M, Barnes PJ, Wongkajornsilp A
J. Allergy Clin. Immunol., 2008-12-05;123(1):239-48.
Species: Human
Sample Types: Whole Cells
Applications: Flow Cytometry

A novel mechanism of CD40-induced apoptosis of carcinoma cells involving TRAF3 and JNK/AP-1 activation.
Authors: Georgopoulos NT, Steele LP, Thomson MJ, Selby PJ, Southgate J, Trejdosiewicz LK
Cell Death Differ., 2006-01-20;13(10):1789-801.
Species: Human
Sample Types: Whole Cells
Applications: Flow Cytometry, Neutralization

The death domain kinase RIP1 links the immunoregulatory CD40 receptor to apoptotic signaling in carcinomas
Authors: Pauline G. Knox, Clare C. Davies, Marina Ioannou, Aristides G. Eliopoulos
Journal of Cell Biology

An orally available small molecule that targets soluble TNF to deliver anti-TNF biologic-like efficacy in rheumatoid arthritis
Authors: Alexander Vugler, James O’Connell, Mai Anh Nguyen, Dietmar Weitz, Thomas Leeuw, Elizabeth Hickford et al.
Frontiers in Pharmacology

FAQs

Does Human TNF-alpha antibody, clone # 6401, detect membrane bound or soluble TNF-alpha?
- Human TNF-alpha antibody, Clone # 6401, binds to and neutralizes Catalog # 210-TA, which is an E. coli-derived human TNF-alpha protein, Val77-Leu233, and has been shown to be cytotoxic to L‑929 mouse fibroblast cells. According to Uniprot, the soluble form of TNF-alpha is amino acids 77-233: https://www.uniprot.org/uniprot/P01375. We have also shown binding of Clone # 6401 to the membrane bound form of TNFa via flow cytometry. We are aware of a few citations showing the antibody acts to neutralize TNFa. See https://www.rndsystems.com/products/human-tnf-alpha-antibody-6401_mab2101#product-citations. Therefore, clone #6401 detects both membrane bound and soluble forms of TNF-alpha.

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