IL12A Antibody (OASA00349)

Catalog#: OASA00349

• Size: 0.5mg

• Description of Target: MOUSE ANTI BOVINE INTERLEUKIN-12:Low Endotoxin
• Gene Symbol: IL12A
• Alias Symbols: IL12p35; Il-12 p35
• Protein Accession#: NP_776780.1
• Swissprot Id: P54349
• Host: Mouse
• Protein Size (# AA): 221
• Specificity: IL-12
• Application: ELISA, FC, WB
• Clone: CC301
• Conjugation: Low Endotoxin
• Immunogen: The immunogen for anti-IL12A antibody: recombinant bovine IL-12
• Product Format: Purified IgG - liquid
• Isotype: IgG2a
• Applications Info: ELISA : This antibody may be used as a capture antibody in a sandwich ELISA in combination with biotinylated clone CC326 (OASA00348) as detection reagent.
  
  Flow Cytometry: Use 10ul of the suggested working dilution to label 10⁶ cells in 100ul.
• Additional Information: Fusion Partners: Spleen cells from immunised BALB/c mice were fused with cells of the mouse SP2/0 myeloma cell line.
  Preparation: Purified IgG prepared by affinity chromatography on Protein G from tissue culture supernatant
  
  Preservative Stabilisers: None present
  Approx Protein Conc: IgG concentration 1.0 mg/ml
  
  Buffer Solutions: Phosphate buffered saline pH7.4
  
  Endotoxin Level: < 0.01 EU/ug
• Reconstitution and Storage: Store at -20^oC only.
  
  This product should be stored undiluted.
  
  Storage in frost free freezers is not recommended. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.
• Key Reference: 1. Hope, J. C. et al. (2002) Development of detection methods for ruminant interleukin (IL) - 12. J. Immunol. Methods. 266: 117 - 126.
  2. Wenz, J.R. et al. (2010) Factors associated with concentrations of select cytokine and acute phase proteins in dairy cows with naturally occurring clinical mastitis. J Dairy Sci. 93: 2458-70.
  3. Rinaldi, M. et al (2010) A sentinel function for teat tissues in dairy cows: dominant innate immune response elements define early response to E. coli mastitis. Funct Integr Genomics. 10: 21-38.

Product Protocols: IL-12p40 and IFN-y protein detection Protocol with IL12A Antibody (Catalog Number: OASA00349)

Product Protocols:
IL-12p40 and IFN-y protein detection Protocol with Gene Name: IL12A Antibody (Catalog Number: OASA00349)

Catalog Number: OASA00349

Reference:
1: Contreras V, Urien C, Guiton R, Alexandre Y, Vu Manh TP, Andrieu T, Crozat K, Jouneau L, Bertho N, Epardaud M, Hope J, Savina A, Amigorena S, Bonneau M, Dalod M, Schwartz-Cornil I.Existence of CD8alpha-like dendritic cells with a conserved functional specialization and a common molecular signature in distant mammalian species.J Immunol.2010 Sep 15;185(6):3313-25.Epub 2010 Aug 1 PubMed PMID: 20702727.

Product Name: IL12A Antibody (OASA00349)

Gene Name: IL12A

Species: Bovine

Sample description: Cytokine that can act as a growth factor for activated T and NK cells, enhance the lytic activity of NK/lymphokine-activated Killer cells, and stimulate the production of IFN-gamma by resting PBMC.

Experiment Name: IL-12p40 and IFN-y protein detection

Experiment Background:
Detection of IL-12p40 protein secretion by immunomagnetically selected CD26+ and CD26- L-DCs (OD at 405 nm in ELISA, three different sheep was performed.
2.IL-12p40 was detected by ELISA using the anti-ruminant CC326 and CC301 mouse mAbs (Serotec, Oxford, U.K.).IFN-y was detected using the anti-ruminant IFN-y CC330 and CC302 mAb (Serotec).
Experiment step:
(i) Vanessa et al.studied the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse).
(ii) This work shows that the heterogeneity of the DC population follows a conserved organization in phylogenetically distant mammals and different tissues, with correspondence between mouse spleen CD8a+/CD11b+ DCs, human blood BDCA3+/BDCA1+ DCs, and sheep skin CD26+/CD26- L-DCs.
(iii) At the protein level on sheep L-DCs, they also found that surface CD205 and secreted IL-12p40 were more expressed by CD26+ L-DCs than by CD26- L-DCs, as is the case for mouse CD8alpha + DCs versus CD11b+ DCs.

Product Protocol: IL12A antibody used to evaluate signals delivered through tcr instruct il-12 receptor (il-12r) expression: il-12 and tumor necrosis factor-alpha synergize for il-12r expression at low antigen dose (OASA00349)

Specificity: IL-12
Product Page: IL12A antibody (OASA00349)
Antibody Pair Available: OASA00349 Capture OASA00348 Detection
Experiment Type: Cell incubations

Protocol:
Medium for incubation of mouse cell cultures consisted of RPMI:EHAA 50:50, with the addition of 2 mM l-glutamine, 100 μg/ml streptomycin, 100 IU/ml penicillin G and 5×10–5 M 2-mercaptoethanol.

Ninety-six-well flat- or round-bottom plates (Costar, Corning, NY) were coated with 200 μl of anti-mouse CD3ε (145-2C11; PharMingen) antibody covering a range of antigen concentrations (1 ng/ml to 3 μg/ml) in PBS for 3 h at 37°C and washed with medium 3–4 times before the addition of naive CD4+ T cells. Anti-mouse CD28 (PharMingen) (1 μg/ml) was added during preincubation with anti-CD3 to coat the plates. Naive CD4+ T cells (3–5× 105/well), incubated at 37°C with exogenous cytokines, were harvested at multiple time points after stimulation: 24 h, 3 days or 5 days to determine the kinetics of IFN-γ and IL-12R mRNA induction. Viability of CD4+ cells was 100% in all cultures under all incubation conditions. Carrier-free IL-12, TNF-α and IL-1α (R & D Systems) were used in cell culture at a concentration of 10 ng/ml. IL-4 was used in cultures at a concentration of 300 U/ml. Purified neutralizing cytokine antibodies (no azide/low endotoxin): anti-IFN (XMG1.2), anti-IL-4 (11B11), anti-IL-12 (C17.8) and anti TNF-α (G281-2626) were obtained from PharMingen, and used at a concentration of 10 μg/ml. DO11.10 CD4 cells (2.5–5 ×106) were incubated with 2–4×106 enriched, irradiated APC in 1.5 ml medium/well in 12-well plates and harvested at the indicated time points. Ovalbumin (OVA) peptide (323–339) (generous gift from Dr William Paul) used to stimulate DO11.10 TgN T cells was added directly to duplicate wells during the incubation.

Summary:
1. No endogenous source of IL-12 was present in cultures and, in addition, exogenous IL-2 was not added to the cultures. Since expression of both IFN-γ and IL-12R is associated with a Th1 phenotype, Jeffrey et al. stimulated naive CD4+ T cells with anti-CD3 (1 μg/ml) for 5.5 days and looked for mRNA expression by semiquantitative RT-PCR. Results were normalized to expression of HPRT and expressed as fold change in mRNA expression relative to anti-CD3 treatment only. 2. The combinations of cytokines TNF-α and IL-12, IL-1α and IL-12, and TNF-α, IL-1α and IL-12 led to demonstrable increases in IFN-γ mRNA expression. IFN-γ mRNA expression was increased from 3.3–4.2 times that of anti-CD3 stimulation only. TNF-α or IL-1α alone did not enhance IFN-γ expression, whereas in this experiment IL-12 alone led to a 3-fold increase in expression.

References:
1: Ahlers JD, Belyakov IM, Matsui S, Berzofsky JA. Signals delivered through TCR instruct IL-12 receptor (IL-12R) expression: IL-12 and tumor necrosis factor-alpha synergize for IL-12R expression at low antigen dose. Int Immunol. 2001 Nov;13(11):1433-42. PubMed PMID: 11675375.