Interferon gamma antibody

Catalog No. ABIN2689586

Product Details anti-Interferon gamma Antibody

Target: Interferon gamma (IFNG)

Reactivity: Human

Host: Mouse

Clonality: Monoclonal

Conjugate: This Interferon gamma antibody is un-conjugated

Application: Immunocytochemistry (ICC)

Brand: BD Pharmingen™

Characteristics: The B27 antibody reacts with human interferon-γ (IFN-γ). The immunogen used to generate the B27 hybridoma was E.coli-expressed recombinant human IFN-γ. This is a neutralizing antibody. The use of B27 antibody for epitope mapping of human IFN-γ has been described. The B27 antibody has been reported not to bind to denatured IFN-γ. Immunocytochemistry: PBMC were isolated from human peripheral blood by density gradient cetrifugation and were cultured for 2 days with plate bound anti-human CD3 and soluble anti-human CD28 in the presence of recombinant human IL-2 and recombinant IL-4. The cells were subsequently harvested, washed, and recultured with recombinant human IL-2 and recombinant human IL-4 for an additional 3 days. Finally, the cells were harvested, washed, and stimulated with PMA (Sigma, 5 ng/mL) and ionomycin (Sigma, 500 ng/mL) in the presence of GolgiStop™ (Cat. No. 554724) for 4 hours at 37 °C. The activated cells were harvested and the level of IFN-γ producing cells were detected by a three-step staining procedure that employs a Biotin Goat anti-mouse IgG secondary antibody (Cat. No. 550337) and Streptavidin-horseradish peroxidase (HRP), Cat. No. 550946. (Nomarski optics, original magnification 400X).

BD Pharmingen™ Purified Mouse Anti-Human IFN-γ - Purified - Clone B27 - Isotype Mouse IgG1, κ - Reactivity Hu - 0.25 mg

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

Immunogen: Human E.coli-expressed IFN-gamma

Clone: B27

Isotype: IgG1 kappa

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 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) 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 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 Cytokine 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 mounding 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-Interferon gamma antibody (ABIN2689586)

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).

Target Details for Interferon gamma

Target: Interferon gamma (IFNG)

Alternative Name: IFN-gamma (IFNG Products)

Synonyms: IFG antibody, IFI antibody, IFN-g antibody, Ifg antibody, IFNG2 antibody, IFN-gamma antibody, IFN-G antibody, IFNG antibody, IFNgamma antibody, TCRalpha antibody, INF-G antibody, ifng antibody, interferon gamma antibody, interferon, gamma 1-2 antibody, IFNG antibody, Ifng antibody, ifng1-2 antibody

Pathways: Interferon-gamma Pathway, 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, ER-Nucleus Signaling, Regulation of Carbohydrate Metabolic Process, Protein targeting to Nucleus, Autophagy