BioPath Online

Pathway Focus: The Interferon Pathway

IFN-induced STAT Phosphorylation
IFN-Induced STAT Phosphorylation—Recombinant Proteins for Interferon Pathway Research
Analyzing Downstream ResponseMeasure Interferon Proteins (IFN-α, IFN-β, and IFN-γ) and Their Downstream Response—Reliable ELISA Kits for Interferon Research

Antibodies for the Interferon Signaling PathwayAntibodies for the Interferon Signaling Pathway—High-Quality, Validated Antibodies
Recombinant Proteins for MAPK Pathway ResearchProfiling Interferon Pathway Biomarkers—Invitrogen™ Multiplex Assays for Luminex® xMAP® Technology
New Antibodies

New Immunoassays

New Molecular Probes® Products

IFN-Induced STAT Phosphorylation—Recombinant Proteins for Interferon Pathway Research

Interferons are involved in various biological processes, including antiviral responses and antiproliferative activities, and are modulators of the immune response. Upon binding to surface receptors, interferons activate the JAK/STAT pathway. We offer a number of recombinant interferons for studying the effects of downstream signaling in the presence of interferons.
The downstream effects of interferon stimulation can be analyzed using the STAT1, 3, 5a/b Phospho 3-Plex kit (Figure 1) or using the ELISA kits described below.

Visit www.invitrogen.com/proteins to view a list of available recombinant interferons and other growth factors.

Analyzing the downstream effects of interferon stimulation

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Figure 1. Analyzing the downstream effects of interferon stimulation using the STAT1, 3, 5a/b Phospho 3-Plex Kit. THP-1 cells were serum-starved overnight, then treated with either 100 ng/mL IFN-γ or 10 ng/mL IFN-α for 15 min or left untreated. Cell lysates were prepared and analyzed with the assay. IFN-α treatment induced phosphorylation of STAT 3 at tyrosine 705, and IFN-γ treatment induced phosphorylation of STAT 1 at tyrosine 701 and STAT 3 at tyrosine 705.



DescriptionQuantity
 Cat. No.
 
Recombinant Human IFN-α A
5 µg
PHC4014
Recombinant Human IFN-γ
100 µg
PHC4031
Recombinant Human IFN-γ
1 mg
PHC4033

Other species of recombinant interferons are also available. Visit www.invitrogen.com/proteins for more information.

Measure Interferon Proteins (IFN-α, IFN-β, and IFN-γ) and Their Downstream Response—Reliable ELISA Kits for Interferon Research

Investigate critical interferon proteins using a reliable method—ELISA (enzyme-linked immunosorbent assay). ELISA is an easy way to measure interferon (IFN) proteins secreted in serum, plasma, or tissue culture supernatant. Using this traditional method of protein quantitation, you can also measure signaling proteins stimulated by interferons in cell lysates.

Invitrogen™ ELISA kits are rigorously validated to ensure excellent quality.
Validation studies include experiments to verify parallelism between calibrated standards and natural samples, peptide competition, and cross-reactivity tests for specificity, and stimulation experiments to ensure correct activation patterns. Specifications include excellent precision (<10% CV), lot-to-lot consistency (<20% CV), recovery (80–120%), and sensitivity (at least 2x more sensitive than western blotting).

Lot-to-lot consistency of IFN-y ELISA Kit

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Figure 2. Lot-to-lot consistency of IFN-γ ELISA Kit.  Individual production lots were analyzed using four levels of control specimens (C1–C4) and ensured low variation between lots (<20%).


Cell Lysate
Dilution
Measured (Units/mL)
Expected (Units/mL)
% Expected
Neat
24.2
24.2
100
1/2
12.5
12.1
103.3
1/4
6.6
6.05
109.3
1/8
3.2
3.03
107.1
1/16
1.78
1.51
117.4

Table 1. STAT3 phosphorylation of Y705 with IFN-α stimulation. HeLa cells were grown in tissue culture medium containing 10% fetal bovine serum, treated with 50 ng/mL IFN-α for 15 min, and lysed with Cell Extraction Buffer. The lysate was diluted in Standard Diluent Buffer over the range of the assay and measured for STAT3 [pY705]. Linear regression analysis of sample values vs. the expected concentration yielded a correlation coefficient of 0.99.


DescriptionSpecies
Quantity Cat. No.

IFN-β Human ELISA Kit
Human
96 tests
KAC1201
Order 
IFN-α Human ELISA Kit
Human
96 tests
KHC4012Order
IFN-γ Human ELISA KitHuman96 testsKHC4021Order
IFN-γ Human ELISA KitHuman192 testsKHC4022Order
STAT3 [pY705] ELISA KitHuman96 testsKHO0481Order

Antibodies for the Interferon Signaling Pathway—High-Quality, Validated Antibodies

The interferon signaling pathway provides a cellular response to viral and other infections. In this mechanism, an infected cell secretes interferon to warn surrounding cells. To keep the infection in check, surrounding cells can effectively commit suicide by shutting down protein synthesis or by proceeding through the apoptosis pathway. This mechanism prevents these cells from becoming virus-producing factories.

There are more than 20 known type I interferons and several type II interferons, and we offer antibodies against most of these targets.  
These Invitrogen™ antibodies have been validated with multiple species using western blotting, ELISA, IHC, and ICC/IF. Learn more at www.invitrogen.com/antibodies.

In addition, we have recently launched the next generation of antibodies, novel ABfinity™ recombinant antibodies. These antibodies also include phosphorylation site–specific antibodies. Learn more about this technology and view a complete list of antibodies at www.invitrogen.com/abfinity.

Immunocytochemistry of HeLa cells labeled with rabbit anti-STAT1 [pY701]

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Figure 4. Immunocytochemistry of HeLa cells labeled with rabbit anti-STAT1 [pY701]. HeLa cells were stimulated with (top right) or without (top left) 100 ng/mL interferon-γ and labeled with rabbit anti-STAT1 [pY701] (1 µg/mL). Stimulated cells were preincubated with the phosphopeptide used as an immunogen (bottom right) or with the  nonphosphopeptide (bottom left), demonstrating phosphospecificity. Alexa Fluor® 488 goat anti–rabbit IgG was used as a secondary antibody at a 1:1,000 dilution.



Western blot of HeLa lysates labeled with rabbit anti-STAT1 [pY701]

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Figure 5. Western blot of HeLa lysates labeled with rabbit anti-STAT1 [pY701]. Rabbit anti-STAT1 [pY701] (0.5 µg/mL) was used to label STAT1 [pY701] in unstimulated HeLa lysates (lane 1) or lysates stimulated with 100 ng/mL interferon-γ (lanes 2–4). Preincubation with the phosphopeptide used as an immunogen eliminated the signal (lane 4), whereas preincubation with the nonphosphopeptide did not (lane 3).


DescriptionReactivity
ApplicationsQuantity
Cat. No.

AKT [pS473] (ABfinity™)
Ms, Hu (Z, X, Rt, Mk (Rh), Mk, Ma, Eq, Fe, Eq, Cp, Ch, Cn, B)
E, F, IHC, IF/ICC, WB
100 µg
700392Order 
STAT1 [pY701] (ABfinity™)
Hu (Or, Mk, B, Sw, Sh, X)
ICC, WB
100 µg
700349Order
IFN-γ
Hu
E, FC, IF, IHC
500 µg
AHC4332Order
IFN-β
Hu
E, IP, IHC, N, WB2 x 104 NU
AHC4024Order
IFN-α A
HuE, IP, IHC, N, WB
1 x 105 NU
AHC4815Order

Reactivity: B= bovine; Ch = chicken; Cn = canine; Cp = chimpanzee; Eq = equine; Fe= feline; Hu = human; Ma = mammalian; Mk (Rh) = monkey (rhesus); Ms = mouse; Or = orangutan; Rt = rat; Sh = sheep; Sw = swine; Z = zebrafish; X = Xenopus. Applications: E = electrophoresis; F = fluorescence; FC = flow cytometry; ICC = immunocytochemistry; IF = immunofluorescence; IHC = immunohistochemistry; IP = immunoprecipitation; N = neutralization studies; WB = western blot.

Profiling Interferon Pathway Biomarkers—Invitrogen™ Multiplex Assays for Luminex® xMAP® Technology

Interferons—proteins that act as antiviral agents—modulate the immune system and play a significant role in cell growth, differentiation, and metabolism. Studying interferons helps clarify how cells and tissues respond to viruses and other infectious agents. IFN-α and IFN-β are considered type I interferons; IFN-γ is classed with type II interferons. These interferon classes are based on the receptor subunits that are used to signal to other proteins.
We offer Singleplex Bead Kits that provide analyte-specific components for measuring IFN-α or IFN-γ in plasma or tissue culture supernatant. The assays may be run separately or in combination with other Invitrogen™ Human Singleplex Bead Kits. These reagents are intended for use with the Luminex® 100™ or 200™ System only.

DescriptionQuantity
Cat. No.

IFN-α Human Singleplex Bead Kit
100 tests
LHC4011Order 
IFN-γ Human Singleplex Bead Kit
100 tests
LHC4031Order
IFN-γ Mouse Singleplex Bead Kit
100 tests
LMC4031Order
IFN-γ Rat Singleplex Bead Kit
100 tests
LRC4031Order
IFN-γ Monkey Singleplex Bead Kit
100 tests
LPC4031Order

For research use only.  Not intended for human or animal therapeutic or diagnostic use.