IFN-γ Antibody [A12D6] | Primary Antibodies

Application: Reactivity: Mouse

Usage Information

Dilution
WB1:1000

Application
WB

Reactivity
Mouse

Source
Rabbit Monoclonal Antibody

Storage Buffer
PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3

Storage (from the date of receipt)
-20°C (avoid freeze-thaw cycles), 2 years

Predicted MW Observed MW
18 kDa 17 kDa, 19 kDa, 23 kDa
^*Why do the predicted and actual molecular weights differ? The following reasons may explain differences between the predicted and actual protein molecular weight. Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.

Datasheet & SDS

Biological Description

Specificity
IFN-γ Antibody [A12D6] detects endogenous levels of total IFN-γ protein.

Clone
A12D6

Synonym(s)
gamma interferon; Ifg; IFN-g; IFN-gamma; Ifng; Interferon gamma; interferon, gamma

Background
Interferon-gamma (IFN-γ) represents the sole type II interferon and functions as a pleiotropic cytokine orchestrating both innate and adaptive immune responses through actions on nearly every cell type except mature erythrocytes. IFN-γ exists as a noncovalently linked homodimer comprising two antiparallel-oriented polypeptide chains each containing six alpha helices forming a compact globular structure with twofold symmetry, with biological activity requiring both amino-terminal residues 1-10 and carboxy-terminal residues 129-143 for full receptor binding competency. The protein undergoes N-glycosylation yielding mature subunits migrating at approximately 25 kDa on SDS-PAGE although glycosylation contributes to proteolytic protection rather than direct functional activity. IFN-γ production becomes largely restricted to T lymphocytes (Th1 CD4+ and cytotoxic CD8+) and natural killer cells, with NK cells maintaining constitutive IFNG locus accessibility enabling rapid secretion during infection or cancer through receptor-mediated activation via immunoreceptor tyrosine-based activating motifs triggering Src/MAPK/ERK/p38 pathways activating Fos and Jun transcription factors, and cytokine-mediated activation primarily through IL-12 binding IL-12 receptors activating STAT4 and NF-κB establishing positive feedback loops favoring Th1 responses. The protein binds a heterodimeric receptor complex composed of IFNGR1 (90 kDa, primary ligand binder) and IFNGR2 (62 kDa, affinity enhancer) with stoichiometry revealing each IFN-γ homodimer engaging two IFNGR1 and two IFNGR2 subunits forming a 2:2:2 signaling-competent complex. IFNGR1 contains constitutive JAK1 binding at membrane-proximal LPKS motif with Pro267 critical for function, plus an inducible STAT1 docking site at YDKPH motif where Tyr440 phosphorylation nucleates STAT1 SH2 domain recruitment, while IFNGR2 harbors constitutive JAK2 binding at PPSIPLQIEEYL sequences. IFN-γ ligation induces IFNGR1-IFNGR2 heterodimerization bringing constitutively receptor-bound but inactive JAK1 and JAK2 into proximity enabling auto- and trans-phosphorylation activating both kinases, with activated JAKs phosphorylating IFNGR1 Tyr440 creating STAT1 docking sites where recruited STAT1 undergoes JAK-mediated Tyr701 phosphorylation plus serine kinase-mediated Ser727 phosphorylation required for complete activation. Phosphorylated STAT1 molecules form homodimers through reciprocal SH2 domain-phosphotyrosine interactions, dissociate from receptors, translocate to the nucleus, and bind gamma-activated site (GAS) DNA elements with consensus TTNCNNNAA sequences, driving transcription of interferon-stimulated genes, including the transcription factors IRF1, IRF2, IRF8, IRF9, and RELA that subsequently activate secondary ISG waves. IFN-γ signaling becomes negatively regulated through SH2-domain-containing phosphatases (SHP proteins) constitutively associated with IFNGR complexes that dephosphorylate activating tyrosine residues, suppressors of cytokine signaling (SOCS) proteins transcriptionally induced by IFN-γ that inhibit and ubiquitinate JAKs for destruction, and protein inhibitors of activated STATs (PIAS) that prevent STAT1-DNA interactions, with nuclear STAT1 dephosphorylation by T-cell protein tyrosine phosphatase TCP45 enabling cytoplasmic recycling. IFN-γ executes pleiotropic functions including activation of macrophages as the major macrophage-activating factor inducing expression of inducible nitric oxide synthase generating microbicidal nitric oxide, production of proinflammatory cytokines IL-1β, TNF, IL-12, IL-18, and IL-23, upregulation of MHC class I and class II molecules plus costimulatory proteins CD80 and CD86 enhancing antigen presentation, induction of immunoproteasome components LMP1, LMP7, and MECL1 altering peptide repertoires, upregulation of TAP-1 and TAP-2 transporters moving peptides into endoplasmic reticulum, and activation of MHC class II transactivator CIITA driving class II expression. The cytokine promotes Th1 differentiation through STAT1 activation of T-bet transcription factor that upregulates IL-12 receptor and IFN-γ expression establishing positive feedback while simultaneously inhibiting Th2 and Th17 proliferation through maintained IFNGR expression rendering these subsets IFN-γ-sensitive, promotes B-cell immunoglobulin class switching from IgE to IgG2a, facilitating antibody-dependent cellular cytotoxicity, enhances CD8+ T-cell cytolytic capacity through upregulation of IL-2 receptor, T-bet, and granzyme expression, and activates NK cell cytotoxicity against tumor cells and infected targets. IFN-γ mediates critical roles in cancer immunoediting, wherein immune selection eliminates tumor cells expressing strong neoantigens—tumors developing in IFN-γ-insensitive IFNGR1-deficient or STAT1-deficient mice arise more frequently and rapidly than wild-type counterparts, with IFN-γ signaling required in both host cells and tumor cells for immune-mediated elimination demonstrated through experiments showing IFN-γ-insensitive tumors grow progressively while IFN-γ-sensitive tumors become rejected following lipopolysaccharide treatment. The protein paradoxically exhibits protumorigenic functions through upregulation of programmed death ligand-1 (PD-L1) and PD-L2 on tumor cells and immune cells enabling adaptive immune resistance, promotion of papilloma development via upregulation of proinflammatory cytokines and Th17 responses, and facilitation of colorectal carcinoma in SOCS1-deficient mice through unchecked IFN-γ signaling. IFN-γ activates additional signaling pathways beyond JAK-STAT including PI3K/Akt/mTOR/p70S6 kinase axis required for translation of interferon-stimulated genes generating antiviral effects, MAPK pathways Pyk2 and ERK1/2, TGFβ/SMAD signaling inducing NADPH oxidases NOX1 and NOX4 generating reactive oxygen species triggering DNA damage and senescence, and STAT1-independent transcription of C/EBPβ and macrophage inflammatory proteins MIP-1α and MIP-1β. The cytokine regulates hematopoiesis with overexpression mediating bone marrow suppression linked to myelodysplastic syndromes and aplastic anemia, exhibits anti-angiogenic effects, participates in neurogenesis and neuronal differentiation via ERK1/2 pathway activation, and controls macrophage metabolism through mTORC1 and MNK kinases converging on translation initiation factor eIF4E.

Background

Interferon-gamma (IFN-γ) represents the sole type II interferon and functions as a pleiotropic cytokine orchestrating both innate and adaptive immune responses through actions on nearly every cell type except mature erythrocytes. IFN-γ exists as a noncovalently linked homodimer comprising two antiparallel-oriented polypeptide chains each containing six alpha helices forming a compact globular structure with twofold symmetry, with biological activity requiring both amino-terminal residues 1-10 and carboxy-terminal residues 129-143 for full receptor binding competency. The protein undergoes N-glycosylation yielding mature subunits migrating at approximately 25 kDa on SDS-PAGE although glycosylation contributes to proteolytic protection rather than direct functional activity. IFN-γ production becomes largely restricted to T lymphocytes (Th1 CD4+ and cytotoxic CD8+) and natural killer cells, with NK cells maintaining constitutive IFNG locus accessibility enabling rapid secretion during infection or cancer through receptor-mediated activation via immunoreceptor tyrosine-based activating motifs triggering Src/MAPK/ERK/p38 pathways activating Fos and Jun transcription factors, and cytokine-mediated activation primarily through IL-12 binding IL-12 receptors activating STAT4 and NF-κB establishing positive feedback loops favoring Th1 responses. The protein binds a heterodimeric receptor complex composed of IFNGR1 (90 kDa, primary ligand binder) and IFNGR2 (62 kDa, affinity enhancer) with stoichiometry revealing each IFN-γ homodimer engaging two IFNGR1 and two IFNGR2 subunits forming a 2:2:2 signaling-competent complex. IFNGR1 contains constitutive JAK1 binding at membrane-proximal LPKS motif with Pro267 critical for function, plus an inducible STAT1 docking site at YDKPH motif where Tyr440 phosphorylation nucleates STAT1 SH2 domain recruitment, while IFNGR2 harbors constitutive JAK2 binding at PPSIPLQIEEYL sequences. IFN-γ ligation induces IFNGR1-IFNGR2 heterodimerization bringing constitutively receptor-bound but inactive JAK1 and JAK2 into proximity enabling auto- and trans-phosphorylation activating both kinases, with activated JAKs phosphorylating IFNGR1 Tyr440 creating STAT1 docking sites where recruited STAT1 undergoes JAK-mediated Tyr701 phosphorylation plus serine kinase-mediated Ser727 phosphorylation required for complete activation. Phosphorylated STAT1 molecules form homodimers through reciprocal SH2 domain-phosphotyrosine interactions, dissociate from receptors, translocate to the nucleus, and bind gamma-activated site (GAS) DNA elements with consensus TTNCNNNAA sequences, driving transcription of interferon-stimulated genes, including the transcription factors IRF1, IRF2, IRF8, IRF9, and RELA that subsequently activate secondary ISG waves. IFN-γ signaling becomes negatively regulated through SH2-domain-containing phosphatases (SHP proteins) constitutively associated with IFNGR complexes that dephosphorylate activating tyrosine residues, suppressors of cytokine signaling (SOCS) proteins transcriptionally induced by IFN-γ that inhibit and ubiquitinate JAKs for destruction, and protein inhibitors of activated STATs (PIAS) that prevent STAT1-DNA interactions, with nuclear STAT1 dephosphorylation by T-cell protein tyrosine phosphatase TCP45 enabling cytoplasmic recycling. IFN-γ executes pleiotropic functions including activation of macrophages as the major macrophage-activating factor inducing expression of inducible nitric oxide synthase generating microbicidal nitric oxide, production of proinflammatory cytokines IL-1β, TNF, IL-12, IL-18, and IL-23, upregulation of MHC class I and class II molecules plus costimulatory proteins CD80 and CD86 enhancing antigen presentation, induction of immunoproteasome components LMP1, LMP7, and MECL1 altering peptide repertoires, upregulation of TAP-1 and TAP-2 transporters moving peptides into endoplasmic reticulum, and activation of MHC class II transactivator CIITA driving class II expression. The cytokine promotes Th1 differentiation through STAT1 activation of T-bet transcription factor that upregulates IL-12 receptor and IFN-γ expression establishing positive feedback while simultaneously inhibiting Th2 and Th17 proliferation through maintained IFNGR expression rendering these subsets IFN-γ-sensitive, promotes B-cell immunoglobulin class switching from IgE to IgG2a, facilitating antibody-dependent cellular cytotoxicity, enhances CD8+ T-cell cytolytic capacity through upregulation of IL-2 receptor, T-bet, and granzyme expression, and activates NK cell cytotoxicity against tumor cells and infected targets. IFN-γ mediates critical roles in cancer immunoediting, wherein immune selection eliminates tumor cells expressing strong neoantigens—tumors developing in IFN-γ-insensitive IFNGR1-deficient or STAT1-deficient mice arise more frequently and rapidly than wild-type counterparts, with IFN-γ signaling required in both host cells and tumor cells for immune-mediated elimination demonstrated through experiments showing IFN-γ-insensitive tumors grow progressively while IFN-γ-sensitive tumors become rejected following lipopolysaccharide treatment. The protein paradoxically exhibits protumorigenic functions through upregulation of programmed death ligand-1 (PD-L1) and PD-L2 on tumor cells and immune cells enabling adaptive immune resistance, promotion of papilloma development via upregulation of proinflammatory cytokines and Th17 responses, and facilitation of colorectal carcinoma in SOCS1-deficient mice through unchecked IFN-γ signaling. IFN-γ activates additional signaling pathways beyond JAK-STAT including PI3K/Akt/mTOR/p70S6 kinase axis required for translation of interferon-stimulated genes generating antiviral effects, MAPK pathways Pyk2 and ERK1/2, TGFβ/SMAD signaling inducing NADPH oxidases NOX1 and NOX4 generating reactive oxygen species triggering DNA damage and senescence, and STAT1-independent transcription of C/EBPβ and macrophage inflammatory proteins MIP-1α and MIP-1β. The cytokine regulates hematopoiesis with overexpression mediating bone marrow suppression linked to myelodysplastic syndromes and aplastic anemia, exhibits anti-angiogenic effects, participates in neurogenesis and neuronal differentiation via ERK1/2 pathway activation, and controls macrophage metabolism through mTORC1 and MNK kinases converging on translation initiation factor eIF4E.

References
https://pubmed.ncbi.nlm.nih.gov/29661791/ https://pubmed.ncbi.nlm.nih.gov/30191398/

Protein Size (kDa)	PVDF Transfer Membrane Pore Size (μm)
< 10	0.22
10-20	0.22
20-30	0.45
30-45	0.45
45-70	0.45
80-100	0.45
100-140	0.45
> 140	0.45

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Protein Size (kDa)	Separating Gel Percentage
4-40	20%
12-45	15%
10-70	12.5%
15-100	10%
25-100	8%
60-210	5%