TNF alpha antibody

Catalog No. ABIN2689933

Product Details anti-TNF alpha Antibody

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Reactivity: Mouse

Host: Rat

Clonality: Monoclonal

Conjugate: This TNF alpha antibody is un-conjugated

Application: Immunocytochemistry (ICC)

Brand: BD Pharmingen™

Characteristics: The MP6-XT22 antibody reacts with mouse tumor necrosis factor (TNF, also known as TNF-α). The immunogen used to generate this hybridoma was recombinant mouse TNF. This antibody is routinely tested by immunocytochemistry. Other applications were tested during antibody development only or reported in the literature. TNF Staining: RBC-lysed BALB/c splenocytes were cultured with PMA (Sigma, 5 ng/mL) and ionomycin (Sigma, 500 ng/mL) with GolgiPlug™ (Cat. No. 555029) for 4 hr at 37 °C. The activated cells were harvested and the presence of TNF producing cells was detected by immunocytochemistry using a three-step staining procedure that employs a Biotin Goat anti-Rat IgG secondary antibody (Cat. No. 559286) and a horseradish peroxidase-based detection system (Nomarski optics, original magnification 400 X). To demonstrate specificity of staining the binding of the MP6-XT22 (Cat. No. 559064) antibody was blocked by the preincubation of the purified antibody with excess recombinant mouse TNF (Cat. No. 554589, data not shown).

BD Pharmingen™ Purified Rat Anti-Mouse TNF - Purified - Clone MP6-XT22 - Isotype Rat IgG1 - Reactivity Ms - 0.25 mg

Purification: The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography.

Immunogen: Recombinant mouse TNF

Clone: MP6

Isotype: IgG1

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Assay Procedure:

FOR IMMUNOCYTOCHEMICAL STAINING OF SINGLE-CELL PREPARATIONS This procedure describes the immunoenzymatic technique of staining cytokines within individual cells that are immobilized on microscopic slides via adherence (adherent slides) or centrifugation (cytospins). ADHESION SLIDES 1. Harvest cells and wash them twice in PBS using centrifugation (400 x g for 5 min) to remove residual protein. 2. Adjust the cell concentration at 4-5 x 10e6 cells/mL in PBS. 3. Place 20 μL of the cell suspension in each well of the adhesion slides and let them adhere at room temperature (RT) for 20 min. Please note that the slides should be washed in PBS at RT for 5 min before transferring the cells. 4. Fix cells on slides using fixation buffer (Cat. No. 550010) for 15 min at RT. 5. Wash slides 2X in PBS with 5 min incubations. 6. Block slides with PBS supplemented with 1 % (w/v) BSA (Sigma, Cat. No. A43-78) for 30 min at RT or 10 min at 37 °C. 7. Wash slides 2X in PBS and proceed with staining or air dry them and store them at -80 °C for future use. 8. Incubate slides with 20 μL of 1 % goat serum and PBS with 0.1 % (w/v) saponin for 30 min at RT. 9. Wash slides 2X with PBS with 5 min incubations. 10. Block endogenous peroxidase activity with Endogenous Peroxidase Blocking Buffer (20 μL/well) for 10 min at RT. 11. Wash 2X in PBS with 5 min incubations. 12. Incubate each well with Avidin (20 μL/well) for 15 min. 13. Wash 2X in PBS with 5 min incubations. 14. Incubate each well with Biotin (20 μL/well) for 15 min. 15. Wash 2X in PBS with 5 min incubations. 16. Incubate each well for 1 hr at RT with 20 μL of purified cytokine-specific antibody or appropriate immunoglobulin isotype control diluted in Pharmingen's IHC Diluent Buffer (Cat. No. 559148), supplemented with saponin. 17. Wash slides 2X in PBS with 5 min incubations. 18. Incubate each well with 20 μL of a biotinylated secondary antibody diluted in IHC Diluent Buffer for 30 min at RT. 19. Wash 2X in PBS with 5 min incubations. 20. Apply 20 μL of Streptavidin-HRP (BD Cat. No. 550946) to each well on slides and incubate for 30 min at RT. 21. Wash slides 2X with PBS with 5 minutes incubations. 22. Incubate with DAB Substrate as directed, (BD Cat. No. 550880) for less than 5 min at RT. 23. Stop the development of the color reaction by washing with PBS. 24. The slides are subsequently mounted in short-term storage mounting medium. CYTOSPINS 1. Assemble the Cytospin's sample chamber (e.g. Cytospin 3, Shandon, UK or comparable centrifuge), filter card, slide and cytospin racks according to manufacturer's specifications. 2. Load 40 μL of approximately 1 x 10e6 cells to each sample chamber. 3. Spin slides at 600 rpm for 2 min. 4. Take slides out of the cytospin rack and place them on a staining rack. 5. For fixation and staining please follow the steps 4 through 24 specified above for staining cells on adhesion slides.

Restrictions: For Research Use only

Handling

Concentration: 0.5 mg/mL

Buffer: Aqueous buffered solution containing ≤0.09 % sodium azide.

Preservative: Sodium azide

Precaution of Use: This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

Storage: 4 °C

Storage Comment: Store undiluted at 4°C.

References for anti-TNF alpha antibody (ABIN2689933)

Abrams: "Immunoenzymetric assay of mouse and human cytokines using NIP-labeled anti-cytokine antibodies." in: Current protocols in immunology / edited by John E. Coligan ... [et al.], Vol. Chapter 6, pp. Unit 6.20, (2008) (PubMed).

Litton, Sander, Murphy, OGarra, Abrams: "Early expression of cytokines in lymph nodes after treatment in vivo with Staphylococcus enterotoxin B." in: Journal of immunological methods, Vol. 175, Issue 1, pp. 47-58, (1994) (PubMed).

Abrams, Roncarolo, Yssel, Andersson, Gleich, Silver: "Strategies of anti-cytokine monoclonal antibody development: immunoassay of IL-10 and IL-5 in clinical samples." in: Immunological reviews, Vol. 127, Issue 9-10, pp. 5-24, (1992) (PubMed).

Hsu, Raine, Fanger: "Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures." in: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, Vol. 29, Issue 4, pp. 577-80, (1981) (PubMed).

Target Details for TNF alpha

Target: TNF alpha (Tumor Necrosis Factor alpha (TNF alpha))

Alternative Name: TNF (TNF alpha Products)

Synonyms: DIF antibody, TNF-alpha antibody, TNFA antibody, TNFSF2 antibody, RATTNF antibody, Tnfa antibody, tnf antibody, TNF-a antibody, TNFalpha antibody, Tnfsf1a antibody, TNFa antibody, cTNF antibody, Tnf-alpha antibody, tnfa-like antibody, TNF-ALPHA antibody, dif antibody, tnfa antibody, xtnf antibody, tnfsf2 antibody, tnf-alpha antibody, Cachectin antibody, tumor necrosis factor antibody, tumor necrosis factor b (TNF superfamily, member 2) antibody, tumor necrosis factor alpha antibody, tumor necrosis factor a (TNF superfamily, member 2) antibody, TNF antibody, Tnf antibody, tnf antibody, tnfb antibody, tnf-alpha antibody, LOC103694380 antibody, tnfa antibody

Pathways: NF-kappaB Signaling, Apoptosis, Caspase Cascade in Apoptosis, TLR Signaling, Cellular Response to Molecule of Bacterial Origin, Regulation of Leukocyte Mediated Immunity, Positive Regulation of Immune Effector Process, Production of Molecular Mediator of Immune Response, Positive Regulation of Endopeptidase Activity, Hepatitis C, Protein targeting to Nucleus, Inflammasome