JAK1 Monoclonal Antibody

What is JAK1 and why is it an important research target?

JAK1 (Janus kinase 1) is a critical tyrosine kinase protein involved in cytokine receptor signaling pathways, cell differentiation, and viral immune responses. In humans, the canonical JAK1 protein consists of 1154 amino acid residues with a molecular weight of approximately 133.3 kDa and is primarily localized in cellular membranes. JAK1 is widely expressed across tissues and can phosphorylate all STAT proteins, making it a central component in multiple signaling cascades. Its aberrant regulation is associated with several pathological conditions including myeloproliferative neoplasms, leukemia, and inflammatory diseases, highlighting its significance as a research and therapeutic target .

How do I select the appropriate JAK1 monoclonal antibody for my specific application?

Selection should be based on several critical parameters:

• Application compatibility: Determine if the antibody is validated for your specific application (Western blot, IHC, ICC, IP, etc.). For example, antibody clone D1T6W is validated for Western blotting (1:1000 dilution) and immunoprecipitation (1:200 dilution) .

• Species reactivity: Verify cross-reactivity with your experimental model organism. Many JAK1 antibodies are reactive against human, mouse, and rat proteins, but specificity varies by clone .

• Epitope recognition: Some antibodies target specific regions, such as the N-terminal domain or specific phosphorylation sites. For example, clone 1I13 targets an epitope within the N-terminal half of JAK1 .

• Validated performance: Check published literature citations and validation data showing specificity in your cell/tissue type of interest .

• Format requirements: Consider whether you need unconjugated or conjugated (HRP, fluorophores) antibodies depending on your detection system .

What is the functional significance of JAK1 phosphorylation sites and how do antibodies targeting these sites differ?

JAK1 activation occurs through phosphorylation of specific tyrosine residues, particularly Tyr1034/1035, which are located in the activation loop. These phosphorylation events alter protein conformation to facilitate substrate binding and initiate downstream signaling .

Antibodies targeting phosphorylated versus non-phosphorylated JAK1 serve distinct research purposes:

When studying signaling dynamics, using both antibody types in parallel can provide insight into both the activation state and expression levels of JAK1, offering a more complete picture of signaling pathway regulation.

What are the optimal sample preparation conditions for detecting JAK1 in different applications?

Successful JAK1 detection requires application-specific sample preparation protocols:

For Western Blotting:

• Use RIPA or NP-40 based lysis buffers containing phosphatase inhibitors (sodium fluoride, sodium orthovanadate) and protease inhibitors

• Maintain samples at 4°C during processing to prevent protein degradation

• Denature samples at 95°C for 5 minutes in reducing conditions (with DTT or β-mercaptoethanol)

• Load 20-50 μg of total protein per well

• Use 7-10% SDS-PAGE gels for optimal resolution of the 130-133 kDa JAK1 protein

For Immunohistochemistry/Immunocytochemistry:

• For paraffin sections, use heat-induced epitope retrieval (HIER) with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

• Optimize fixation time (10% neutral buffered formalin for 24-48 hours is typical)

• Block endogenous peroxidase activity using hydrogen peroxide

• For immunocytochemistry, a 1:100 dilution has been validated for certain antibodies

For Immunoprecipitation:

• Use milder lysis buffers (e.g., 1% NP-40, 150 mM NaCl, 50 mM Tris pH 8.0)

• A typical dilution of 1:200 works well for some JAK1 antibodies

• Pre-clear lysates with protein A/G beads to reduce non-specific binding

How can I optimize JAK1 antibody-based detection methods for low-abundance samples?

When working with samples where JAK1 expression is limited, consider these optimization strategies:

• Signal amplification methods:

  • Use high-sensitivity ECL substrates for Western blots

  • Consider tyramide signal amplification (TSA) for IHC/ICC applications

  • Employ biotin-streptavidin systems for enhanced signal

• Sample enrichment techniques:

  • Increase protein concentration through immunoprecipitation before Western blotting

  • Use phospho-enrichment methods if studying activated JAK1

  • Consider cell fractionation to concentrate membrane fractions where JAK1 is predominantly localized

• Antibody optimization:

  • Extend primary antibody incubation time (overnight at 4°C)

  • Optimize antibody concentration through titration experiments

  • Use higher antibody concentrations than standard protocols (with proper controls to assess specificity)

• Detection system selection:

  • For Western blots, use fluorescent secondary antibodies and digital imaging systems

  • For microscopy, consider confocal imaging with photomultiplier detectors set for maximum sensitivity

How can I design experiments to study JAK1 interactions with different cytokine receptors?

To investigate JAK1 interactions with specific cytokine receptors:

• Co-immunoprecipitation (Co-IP) approaches:

  • Immunoprecipitate JAK1 using validated antibodies (e.g., clone D1T6W at 1:200 dilution)

  • Probe for co-precipitated receptors (γc receptor, gp130, IFN receptors) with specific antibodies

  • Alternatively, immunoprecipitate the receptor and probe for JAK1

  • Use crosslinking reagents for transient interactions

• Proximity ligation assays (PLA):

  • Apply primary antibodies against JAK1 and the receptor of interest

  • Use PLA probes and detection reagents to visualize interactions in situ

  • Quantify interaction signals in different cellular compartments

• Receptor stimulation experiments:

  • Treat cells with specific cytokines (IL-6, IFNs, IL-2 family)

  • Monitor JAK1 phosphorylation status and receptor association at different time points

  • Compare wild-type vs. receptor mutants to map interaction domains

• CRISPR-based approaches:

  • Generate JAK1 domain mutants to identify regions necessary for specific receptor interactions

  • Assay signaling outcomes downstream of different cytokine receptors

What are common causes of non-specific binding with JAK1 antibodies and how can they be addressed?

Non-specific binding is a frequent challenge when working with JAK1 antibodies. Common causes and solutions include:

Causes and Solutions:

• Cross-reactivity with related JAK family members:

  • JAK1 shares structural homology with JAK2, JAK3, and TYK2

  • Solution: Verify antibody specificity using knockout/knockdown controls or JAK-specific inhibitors

  • Use antibodies targeting unique epitopes (e.g., N-terminal regions)

• High background in immunostaining:

  • Caused by insufficient blocking or non-specific secondary antibody binding

  • Solution: Extend blocking time (2+ hours), increase blocking agent concentration (5% BSA/normal serum)

  • Include 0.1-0.3% Triton X-100 in blocking buffer for permeabilized samples

  • Use species-specific secondary antibodies pre-adsorbed against other species

• Multiple bands in Western blots:

  • May represent post-translational modifications, degradation products, or non-specific binding

  • Solution: Include positive control lysates with known JAK1 expression

  • Optimize sample preparation to prevent protein degradation

  • Verify molecular weight (expected ~130-133 kDa for full-length JAK1)

• Inconsistent results between experiments:

  • Solution: Standardize protocols, particularly antibody dilution and incubation conditions

  • Prepare larger batches of antibody dilutions to use across multiple experiments

  • Document lot numbers, as antibody performance can vary between production batches

How can I accurately interpret phospho-JAK1 signals in the context of pathway activation?

Interpreting phospho-JAK1 signals requires careful consideration of several factors:

• Temporal dynamics:

  • JAK1 phosphorylation typically occurs rapidly (within minutes) after cytokine stimulation

  • Include multiple time points in stimulation experiments (0, 5, 15, 30, 60 minutes)

  • Consider that different cytokines may induce distinct phosphorylation kinetics

• Signal normalization:

  • Always normalize phospho-JAK1 signals to total JAK1 levels

  • Account for loading controls (β-actin, GAPDH) to ensure equal protein loading

  • Present data as phospho/total JAK1 ratios for accurate pathway activation assessment

• Pathway context:

  • JAK1 functions within complexes with other JAKs and cytokine receptors

  • Monitor downstream STAT phosphorylation to confirm functional pathway activation

  • Specific STATs are preferentially activated by different cytokine-receptor-JAK complexes

• Feedback regulation:

  • SOCS proteins provide negative feedback by inhibiting JAK activity

  • Sustained stimulation may show reduced phospho-JAK1 signals due to feedback inhibition

  • Consider measuring SOCS expression alongside JAK1 phosphorylation

What controls should be included when validating JAK1 antibody specificity?

Proper validation of JAK1 antibody specificity requires comprehensive controls:

• Positive controls:

  • Cell lines with verified JAK1 expression (e.g., HeLa, C6, COS-7, Ramos)

  • Recombinant JAK1 protein (full-length or fragments depending on epitope)

  • Tissues known to express JAK1 (e.g., colon, kidney)

• Negative controls:

  • JAK1 knockout or knockdown samples (CRISPR-Cas9 or siRNA)

  • JAK1-null cell lines if available

  • Secondary antibody-only controls to assess background

  • Isotype controls matching the host species and isotype of the JAK1 antibody

• Specificity controls:

  • Peptide competition assays using the immunizing peptide

  • Cross-reactivity assessment with other JAK family members

  • Multiple antibodies targeting different JAK1 epitopes should yield consistent results

  • Affinity binding assays with known KD values (e.g., KD of 1.9 x 10^-7 for clone 1I13)

• Application-specific controls:

  • For phospho-specific antibodies: treatment with phosphatase

  • For Western blots: molecular weight markers to confirm expected size (130 kDa)

  • For IHC/ICC: gradient of expression across different tissues/cells with varying JAK1 levels

How can JAK1 monoclonal antibodies be utilized to investigate chromatin remodeling and non-canonical JAK/STAT signaling?

Recent research has uncovered non-canonical JAK/STAT signaling involving nuclear functions and chromatin remodeling. JAK1 antibodies can be instrumental in studying these processes:

• Chromatin Immunoprecipitation (ChIP) applications:

  • Use JAK1 antibodies to immunoprecipitate chromatin-associated JAK1

  • Sequence associated DNA (ChIP-seq) to identify genomic binding sites

  • Combine with STAT ChIP to correlate JAK1 and STAT binding patterns

• Investigation of heterochromatin association:

  • Perform co-immunoprecipitation of JAK1 with heterochromatin protein-1 (HP1)

  • Use JAK1 antibodies in proximity ligation assays with HP1 to visualize interactions

  • Monitor heterochromatin stability through JAK1 manipulation

• Subcellular fractionation studies:

  • Use JAK1 antibodies to detect nuclear vs. cytoplasmic JAK1 pools

  • Examine how cytokine stimulation affects JAK1 nuclear translocation

  • Investigate association with nuclear proteins like histones or transcription factors

• Experimental approaches:

  • Compare conventional vs. atypical JAK/STAT signaling using JAK inhibitors

  • Examine effects of IFN stimulation on MHC chromosomal remodeling

  • Study JAK3-STAT5 effects on chromatin remodeling at the Ifng locus during Th1 differentiation

What methodologies can be employed to study JAK1 regulation by SOCS proteins using monoclonal antibodies?

SOCS proteins are critical negative regulators of JAK signaling. These methodologies can help elucidate JAK1-SOCS interactions:

• Co-immunoprecipitation strategies:

  • Immunoprecipitate JAK1 and probe for associated SOCS1/SOCS3

  • Perform reverse co-IP using SOCS antibodies and detect JAK1

  • Study how cytokine stimulation timing affects JAK1-SOCS complex formation

• Ubiquitination assays:

  • Immunoprecipitate JAK1 under denaturing conditions

  • Probe for ubiquitin to assess SOCS-mediated ubiquitination

  • Use proteasome inhibitors to enhance detection of ubiquitinated JAK1 species

• Structure-function analysis:

  • Examine how the SOCS3 SH2 domain interacts with phosphorylated JAK1

  • Study how SOCS1 KIR domain inhibits JAK1 kinase activity

  • Investigate cullin5-elongin B/C-SOCS E3 ligase complex formation

• Functional regulation studies:

  • Monitor JAK1 phosphorylation kinetics in cells with varying SOCS expression

  • Examine how SOCS3 regulates gp130-JAK1 signaling

  • Investigate how SOCS proteins differentially regulate JAK1 vs. other JAK family members

How can JAK1 antibodies be applied to investigate differential cytokine receptor signaling networks?

JAK1 is uniquely positioned at the nexus of multiple cytokine receptor families. To dissect these networks:

• Receptor complex immunoprecipitation:

  • Use JAK1 antibodies to pull down receptor complexes from cells stimulated with different cytokines

  • Identify co-precipitated proteins by mass spectrometry or targeted Western blotting

  • Compare receptor associations across different cell types and stimulation conditions

• Phospho-mapping techniques:

  • Use phospho-specific JAK1 antibodies to examine activation in response to different cytokines

  • Examine phosphorylation kinetics of JAK1 vs. downstream STATs

  • Investigate how receptor-specific JAK1 activation leads to different STAT activation patterns

• Multiplexed signaling analysis:

  • Employ multiplexed phospho-flow cytometry with JAK1 antibodies

  • Simultaneously measure JAK1, receptor, and STAT phosphorylation

  • Develop computational models of receptor-specific signaling networks

• Specific receptor systems to investigate:

  • γc receptor family (IL-2, IL-4, IL-7, IL-9, IL-15)

  • Class II cytokine receptors (IFNα/β, IFN-γ, IL-10 family)

  • gp130 receptor family (IL-6, IL-11, CNTF, OSM, LIF, CT-1)

How are JAK1 monoclonal antibodies being utilized in studying inflammatory disease mechanisms and therapeutic development?

JAK1 plays a central role in inflammatory signaling, making JAK1 antibodies valuable tools in inflammation research:

• Inflammation pathway dissection:

  • Use JAK1 antibodies to monitor activation status in inflammatory disease tissues

  • Correlate JAK1 phosphorylation with inflammatory cytokine production

  • Examine cell type-specific JAK1 activation in complex inflammatory microenvironments

• Therapeutic mechanism studies:

  • Investigate how JAK inhibitors affect JAK1 phosphorylation status and receptor association

  • Monitor changes in JAK1-STAT pathway activation in patient samples before and after JAK inhibitor treatment

  • Study how JAK inhibitors reduce IL-6 or IFN-γ serum levels through JAK1 inhibition

• Experimental disease models:

  • Apply JAK1 antibodies in models of inflammatory bowel disease, rheumatoid arthritis, and psoriasis

  • Examine tissue-specific JAK1 activation dynamics during disease progression

  • Correlate JAK1 phosphorylation status with therapeutic outcomes

• Biomarker development:

  • Assess phospho-JAK1 as a potential biomarker for inflammatory disease activity

  • Evaluate JAK1 expression/phosphorylation as predictors of therapeutic response

  • Develop phospho-JAK1 assays suitable for clinical laboratory applications

What approaches can be used to study JAK1 conformational changes and interaction with inhibitors?

Understanding JAK1 structure-function relationships is critical for inhibitor development:

• Structural biology approaches:

  • Use conformation-specific antibodies to detect distinct JAK1 states

  • Employ antibodies to stabilize specific JAK1 conformations for structural studies

  • Develop antibodies that compete with inhibitor binding to map binding sites

• Drug-protein interaction studies:

  • Design competition assays between JAK1 antibodies and small molecule inhibitors

  • Use JAK1 antibodies in cellular thermal shift assays (CETSA) to assess inhibitor engagement

  • Combine with hydrogen-deuterium exchange mass spectrometry to map conformational changes

• Resistance mechanism investigation:

  • Apply antibodies to detect JAK1 mutations associated with inhibitor resistance

  • Examine how resistance mutations affect JAK1 conformation using epitope-specific antibodies

  • Study altered protein-protein interactions in resistant JAK1 variants

• Advanced microscopy techniques:

  • Use Förster resonance energy transfer (FRET) between labeled antibodies and JAK1 to detect conformational changes

  • Apply super-resolution microscopy to visualize JAK1 clustering at the membrane

  • Develop live-cell reporters based on JAK1 antibody fragments

How can JAK1 antibodies contribute to understanding the role of JAK1 in tumor microenvironment and cancer therapy resistance?

JAK1 signaling plays complex roles in cancer biology:

• Tumor microenvironment analysis:

  • Use multiplex immunofluorescence with JAK1 antibodies to map signaling in different cell populations

  • Examine JAK1 activation in tumor-associated immune cells vs. cancer cells

  • Correlate JAK1 expression with tumor immune infiltration patterns

• Therapy resistance studies:

  • Compare JAK1 expression/phosphorylation in therapy-sensitive vs. resistant tumors

  • Investigate JAK1 mutations that contribute to immunotherapy resistance

  • Examine how JAK1 signaling affects PD-L1 expression and anti-tumor immunity

• Experimental approaches:

  • Use patient-derived xenografts to study JAK1 signaling in human tumors

  • Apply single-cell techniques with JAK1 antibodies to resolve heterogeneity

  • Develop JAK1 activity reporters for live imaging in tumor models

• Clinical correlations:

  • Analyze JAK1 expression in colon tumors, where it's expressed at higher levels than normal tissue

  • Investigate JAK1 as a predictive biomarker for immunotherapy response

  • Study JAK1 inactivating mutations in the context of immunotherapy resistance