TNF-α Antibody, anti-human

Name: TNF-α Antibody, anti-human
Availability: InStock

TNF-α Antibody, anti-humanExtended Validation

Clone:

cA2

Type of antibody:

Primary antibodies, Functional-grade antibodies

Isotype:

human IgG1

Applications:

ICFC, MICS, IF, IHC, FA

Alternative names:

TNF, DIF, TNFSF2

Intracellular flow cytometry applications forTNF-α Antibody, anti-human

APC
APC-Vio 770
FITC
PE
PE-Vio 770

Conjugate:

APC

Recommended dilution:

1:50

Remark:

The antibody is suited for staining of formaldehyde-fixed cells.

Tested:

QC tested

Products used:

130-117-531, 130-117-382

Protocols:

Intracellular flow cytometry staining protocol (Inside Stain Kit 1:50)

Description:

Human peripheral blood mononuclear cells (PBMCs) were either left unstimulated (left images) or stimulated with SEB for six hours. After two hours, brefeldin A was added. The cells were fixed, permeabilized, and intracellularly stained with Anti-TNF-α antibodies as well as with CD4 antibodies and CD154 antibodies. Cells were then analyzed by flow cytometry using the MACSQuant^® Analyzer. The Tandem Signal Enhancer has been used to increase binding specificity of tandem-dye–conjugated antibodies. CD4⁺ lymphocets were pre-gated for the analysis. Cell debris were excluded from the analysis based on scatter signals.

MICS (MACSima Imaging Cyclic Staining) applications forTNF-α Antibody, anti-human

APC
FITC
PE

Conjugate:

APC

Recommended dilution:

1:50

Fixation:

Acetone

Tested:

during development

Species tested:

human

Products used:

130-117-531, 130-117-382

Compatible applications:

IF, IHC

Protocols:

Protocol for MICS experiments

Functional assay applications forTNF-α Antibody, anti-human

pure-functional grade

Conjugate:

pure-functional grade

Tested:

during development

Products used:

130-095-749

Type of functional assay:

neutralization of cytokine activity

Extended validation for TNF-α Antibody, anti-human

Specificity

Epitope competition

In order to compare the epitope specificity of an antibody, the clone being used is compared with other known clones recognizing the same antigen in a competition assay.

Other clones	Overlap in epitope recognition with cA2
REA656	++
mab11	++
357-167-24	-
Cells were incubated with an excess of purified unconjugated TNF-α (cA2) antibody followed by staining with fluorochrome-conjugated antibodies of other known clones against the same marker. Based on the fluorescence signal obtained, the clones were identified as recognizing completely overlapping (++), partially overlapping (+), or completely different epitopes (-) of the marker.

Sensitivity

Performance comparison

Selected fluorochrome conjugated antibodies from Miltenyi Biotec were compared to commercially available hybridoma clones in flow cytometry analysis.

Flow cytometric comparison of different clones for TNF-α. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL brefeldin A for 4 hours. Cells were stained with a suitable counterstaining and then fixed and permeabilized followed by a staining with TNF-α antibodies. As a control, TNF-α antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD3⁺/CD4⁺ cells were pregated for the analysis. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for TNF-α. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL brefeldin A for 4 hours. Cells were stained with a suitable counterstaining and then fixed and permeabilized followed by a staining with TNF-α antibodies. As a control, TNF-α antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD3⁺/CD4⁺ cells were pregated for the analysis. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for TNF-α. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL brefeldin A for 4 hours. Cells were stained with a suitable counterstaining and then fixed and permeabilized followed by a staining with TNF-α antibodies. As a control, TNF-α antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD3⁺/CD4⁺ cells were pregated for the analysis. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Flow cytometric comparison of different clones for TNF-α. Human peripheral blood mononuclear cells (PBMCs) were stimulated with 20 ng/mL Phorbol 12-myristate 13-acetate (PMA) and 1 µg/ml Ionomycin for 1 hour, followed by an incubation with 1µg/mL brefeldin A for 4 hours. Cells were stained with a suitable counterstaining and then fixed and permeabilized followed by a staining with TNF-α antibodies. As a control, TNF-α antibody staining was omitted and cells were measured in the same channels. Flow cytometry was performed with the MACSQuant^® Analyzer. CD3⁺/CD4⁺ cells were pregated for the analysis. Cell debris and cell doublets were excluded from the analysis based on scatter signals. No FcR Blocking Reagent was used. The recommended titers of respective antibodies from different suppliers were used.

Specifications for TNF-α Antibody, anti-human

Overview

Tumor necrosis factor alpha (TNF-α) is produced by cells that are involved in inflammatory immune responses. TNF-α is secreted by activated CD4

⁺

T cells, monocytes, macrophages, NK cells, and neutrophils.

Anti-TNF-α antibodies have been designed for intracellular staining of TNF-α–producing cells. Cells can be stimulated for TNF-α production, for example, by polyclonal stimulation with mitogens. For induction of TNF-α production by antigen-specific T cells, cells are restimulated with the respective antigen. TNF-α can be accumulated in the cells by addition of secretion inhibitors like brefeldin A. After fixation and permeabilization of the cell sample, TNF-α–producing cells can be stained intracellularly with Anti-TNF-α antibodies. Staining for surface markers allows simultaneous flow cytometric analysis of subsets and activation status of the TNF-α–producing cells.

Alternative names

TNF, DIF, TNFSF2

Detailed product information

Technical specifications

Clone	cA2
Clonality	monoclonal
Isotype	human IgG1
Host	human
Type of antibody	Primary antibodies, Functional-grade antibodies
Species	human
Cross-reactivity	rhesus monkey ( Macaca mulatta ) , cynomolgus monkey ( Macaca fascicularis )
Antigen	TNF-α
Alternative names of antigen	TNF, DIF, TNFSF2
Molecular mass of antigen [kDa]	26
Entrez Gene ID	7124
RRID	AB_2752116, AB_2752165, AB_2752115, AB_2751396, AB_2751384, AB_2784484, AB_2784483, AB_2752167, AB_2752117, AB_10839558, AB_2752166

Resources for TNF-α Antibody, anti-human

Certificates

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link

to search for Certificates of Analysis (CoA) by lot number.

Applications

Extended validation