Phospho-JAK2 (Tyr1007) Antibody

What is JAK2 and what is the significance of phosphorylation at Tyr1007/1008 sites?

JAK2 (Janus kinase 2) is a protein tyrosine kinase involved in cytokine receptor signaling pathways. The phosphorylation of tyrosine residues 1007/1008 in the activation loop of JAK2 is critical for regulating its kinase activity. These residues are homologous to tyrosine residues 1054/1055 in Tyk2, which play similar regulatory roles in Tyk2 kinase activity . Phosphorylation at these sites occurs during cytokine signaling and leads to downstream activation of STAT (signal transducers and activators of transcription) proteins, which ultimately regulate gene transcription including interferon-stimulated genes .

How does JAK2 function within the JAK-STAT signaling pathway?

In the JAK-STAT pathway, JAK2 functions as follows:

• JAK2 associates with cytokine receptors that lack intrinsic kinase activity

• Upon cytokine binding to receptors, JAK2 becomes activated through auto-phosphorylation at Tyr1007/1008

• Activated JAK2 phosphorylates the cytokine receptor, creating docking sites for STAT proteins

• STAT proteins are recruited and phosphorylated by JAK2

• Phosphorylated STATs dimerize and translocate to the nucleus

• In the nucleus, STAT dimers regulate transcription of target genes

JAK2 is particularly important for IFN-gamma receptor signaling, where it initiates the signaling cascade while JAK1 functions as an amplifier . Research has suggested that Tyk2 and JAK3 might potentially substitute for JAK2 function if properly positioned within the IFN-gamma receptor complex .

What applications are phospho-JAK2 (Tyr1007/1008) antibodies validated for?

Based on the technical information provided by manufacturers, phospho-JAK2 (Tyr1007/1008) antibodies are validated for multiple applications:

For optimal results, researchers should validate these dilutions in their specific experimental systems .

How should samples be prepared to preserve JAK2 phosphorylation status?

To maintain JAK2 phosphorylation during sample preparation:

• Rapidly extract proteins using ice-cold lysis buffers containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

• For tissue samples, flash-freeze in liquid nitrogen immediately after collection

• Use detergent-based lysis buffers (e.g., RIPA or NP-40) with protease inhibitors

• Maintain samples at 4°C throughout processing

• Avoid repeated freeze-thaw cycles of protein lysates

• Process samples quickly to minimize time for phosphatase activity

• If using cultured cells, stimulate with appropriate cytokines just before lysis to maximize phosphorylation signals

These practices help prevent artificial dephosphorylation during sample handling .

What are the optimal conditions for detecting phospho-JAK2 by Western blotting?

For optimal Western blot detection of phospho-JAK2 (Tyr1007/1008):

• Protein concentration: Load 20-50 μg of total protein per lane

• Gel percentage: Use 7-8% SDS-PAGE gels (JAK2 is approximately 125-130 kDa)

• Transfer conditions: Transfer proteins to PVDF membranes at 30V overnight at 4°C

• Blocking: Block membranes with 5% BSA in TBST (not milk, which contains phosphatases)

• Primary antibody: Dilute phospho-JAK2 antibody 1:1000 in 5% BSA/TBST and incubate overnight at 4°C

• Washing: Wash membranes 3-4 times with TBST, 5-10 minutes each

• Secondary antibody: Use appropriate HRP-conjugated secondary antibody at 1:2000-1:5000

• Detection: Use enhanced chemiluminescence (ECL) detection systems

Manufacturers recommend these specific dilutions:

• Cell Signaling Technology antibody #3771: 1:1000 dilution

• Cell Signaling Technology antibody #3776: 1:1000 dilution

• Bio-Techne antibody #NBP3-21579: 1:500-1:2000 dilution

What controls should be included when using phospho-JAK2 (Tyr1007/1008) antibodies?

Proper experimental controls are essential:

• Positive controls:

  • Cell lines treated with cytokines known to activate JAK2 (e.g., IFN-γ, IL-6, growth hormone)

  • Specialized positive control lysates (available from some manufacturers)

• Negative controls:

  • Unstimulated cells (showing baseline phosphorylation)

  • JAK2 inhibitor-treated cells (e.g., AG490, ruxolitinib)

  • siRNA/shRNA JAK2 knockdown samples

• Specificity controls:

  • Blocking with immunizing phosphopeptide

  • Probing parallel blots with total JAK2 antibody to confirm protein presence

  • Treatment with lambda phosphatase to remove phosphorylation

• Loading controls:

  • Probing for housekeeping proteins (β-actin, GAPDH)

  • Total protein stains (Ponceau S, REVERT total protein stain)

These controls help validate antibody specificity and experimental reliability .

How do phospho-JAK2 antibodies contribute to understanding JAK2 mutation-associated diseases?

Phospho-JAK2 antibodies provide critical insights into diseases associated with JAK2 mutations:

• Myeloproliferative neoplasms (MPNs):

  • Detecting constitutive JAK2 activation in polycythemia vera patients with JAK2 V617F mutation

  • Monitoring phosphorylation status before and after JAK inhibitor treatment

  • Quantifying aberrant JAK2 signaling in primary patient samples

• Research applications:

  • Evaluating efficacy of novel JAK2 inhibitors through phosphorylation status

  • Studying resistance mechanisms to JAK2 inhibitor therapy

  • Investigating downstream signaling pathways affected by mutant JAK2

• Clinical correlations:

  • Assessing correlation between JAK2 phosphorylation levels and disease severity

  • Monitoring therapy response through phospho-JAK2 levels

These antibodies enable researchers to study diseases including polycythemia vera, thrombocythemia, myelofibrosis, and acute myelogenous leukemia where JAK2 plays a pathogenic role .

What methodologies can be used to study JAK2 signaling dynamics?

To investigate JAK2 signaling dynamics:

• Time-course experiments:

  • Stimulate cells with cytokines and collect samples at various timepoints (30 seconds to 24 hours)

  • Use phospho-JAK2 antibodies to track activation kinetics

  • Correlate with downstream STAT phosphorylation patterns

• Spatial distribution analysis:

  • Employ immunofluorescence with phospho-JAK2 antibodies to track subcellular localization

  • Use co-localization studies with receptor molecules to examine signaling complexes

  • Apply high-resolution microscopy techniques (confocal, TIRF) for detailed spatial information

• Quantitative approaches:

  • Phospho-flow cytometry for single-cell analysis of JAK2 activation

  • Multiplexed analysis of JAK2 and downstream targets (STAT3, STAT5)

  • Mass cytometry (CyTOF) for comprehensive signaling network analysis

These approaches provide detailed insights into the temporal and spatial dynamics of JAK2 signaling .

What are common issues with phospho-JAK2 antibodies and how can they be resolved?

Researchers frequently encounter these challenges:

• Weak or absent signal:

  • Increase antibody concentration or incubation time

  • Ensure proper stimulation of cells (e.g., with IFN-γ, IL-6)

  • Try alternative detection methods with higher sensitivity

  • Verify phosphatase inhibitors were included in lysis buffer

  • Check for rapid processing of samples to prevent dephosphorylation

• High background:

  • Increase blocking time or concentration (5% BSA recommended)

  • Use more stringent washing steps (longer, more frequent)

  • Decrease antibody concentration

  • Try alternative blocking agents (casein, commercial blockers)

  • Ensure proper antibody dilution in 5% BSA (not milk)

• Non-specific bands:

  • Verify antibody specificity with appropriate controls

  • Optimize primary antibody concentration

  • Consider using alternative clone if available

  • Pre-clear lysates with protein A/G beads before immunoprecipitation

• Inconsistent results:

  • Standardize lysate preparation protocols

  • Maintain consistent stimulation parameters

  • Use freshly prepared reagents

  • Avoid repeated freeze-thaw cycles of antibodies and samples

These solutions are based on technical information from manufacturers and research experience .

How can phospho-JAK2 detection be optimized in different cell types and tissues?

Optimizing phospho-JAK2 detection across different experimental systems:

• Primary cells vs. cell lines:

  • Primary cells may require gentler lysis conditions

  • Cell lines often show higher basal phosphorylation

  • Adjust stimulation protocols (duration, concentration) for different cell types

  • Consider cell-specific JAK2 expression levels when planning experiments

• Tissue-specific considerations:

  • For immunohistochemistry, optimize antigen retrieval methods (heat vs. enzymatic)

  • Different tissues may require adjusted fixation protocols

  • Consider tissue-specific phosphatase activity and adjust inhibitor cocktails

  • For brain tissue, extend fixation time; for liver, enhance permeabilization

• Species-specific optimization:

  • Verify cross-reactivity with your species of interest

  • The phospho-JAK2 (Tyr1007/1008) epitope is conserved across human, mouse, and rat

  • Some antibodies show reactivity only with specific species (check manufacturer specifications)

• Protocol adjustments:

  • For flow cytometry: optimize fixation/permeabilization protocols

  • For immunofluorescence: adjust detergent type and concentration for cell type

  • For Western blotting: modify gel percentage based on tissue-specific protein content

These considerations help maximize signal-to-noise ratio in different experimental systems .

How can phospho-JAK2 antibodies be utilized to investigate inflammatory and immune disorders?

Phospho-JAK2 antibodies offer valuable insights into inflammatory and immune conditions:

• Cytokine signaling analysis:

  • Evaluate JAK2 activation in response to pro-inflammatory cytokines

  • Correlate phospho-JAK2 levels with disease severity in animal models

  • Track changes in JAK2 activation during disease progression

• Therapeutic evaluation:

  • Monitor JAK inhibitor efficacy through phospho-JAK2 reduction

  • Compare different JAK inhibitors' potency and selectivity

  • Investigate resistance mechanisms to JAK inhibitor therapy

• Cell-specific responses:

  • Use flow cytometry with phospho-JAK2 antibodies to identify responsive immune cell subsets

  • Analyze differential JAK2 activation across cell populations

  • Correlate with functional outcomes (cytokine production, proliferation)

These approaches have been applied to study conditions including rheumatoid arthritis, inflammatory bowel disease, and autoimmune disorders where JAK-STAT signaling plays important roles .

What are the technical considerations for analyzing phospho-JAK2 in primary patient samples?

Working with clinical specimens requires special attention:

• Sample preservation:

  • Process samples immediately after collection

  • Use specialized fixatives that preserve phospho-epitopes

  • Consider using phosphatase inhibitor cocktails during collection

• Protocol modifications:

  • Optimize lysis buffers for specific tissue types

  • Adjust antibody concentrations for potentially lower target abundance

  • Consider sensitivity limitations with precious samples

• Analytical approaches:

  • Use quantitative methods (ELISA, phospho-flow) for objective measurement

  • Include appropriate normal tissue controls

  • Normalize phospho-JAK2 signals to total JAK2 levels

• Clinical correlation methodology:

  • Establish standardized scoring systems for immunohistochemistry

  • Use digital pathology tools for quantitative assessment

  • Correlate phospho-JAK2 levels with clinical parameters and outcomes

These considerations help ensure reliable phospho-JAK2 detection in complex clinical samples .

How do monoclonal and polyclonal phospho-JAK2 antibodies compare in research applications?

When selecting between antibody formats:

Researchers should select the appropriate antibody format based on their specific application requirements .

What are the advantages of using recombinant antibodies for phospho-JAK2 detection?

Recombinant phospho-JAK2 antibodies offer several benefits:

• Technical advantages:

  • Superior lot-to-lot consistency

  • Defined sequence eliminates batch variation

  • Animal-free manufacturing process

  • Potentially higher specificity due to controlled production

• Experimental benefits:

  • More reproducible results across experiments

  • Reliable supply without animal source limitations

  • Often available in multiple formats (unconjugated, various conjugates)

  • Can be engineered for specific properties (increased affinity, stability)

• Available options:

  • Cell Signaling Technology offers recombinant rabbit mAb #3776 (C80C3 clone)

  • Bio-Techne provides recombinant monoclonal rabbit antibody NBP3-21579 (SR1129 clone)

  • Invitrogen's phospho-JAK2 recombinant rabbit monoclonal antibody JAK2Y10071008-PB6

These advantages make recombinant antibodies increasingly preferred for critical research applications requiring high reproducibility .

How might phospho-JAK2 antibodies contribute to emerging single-cell analysis techniques?

Phospho-JAK2 antibodies are poised to advance single-cell research through:

• Integration with cutting-edge technologies:

  • Single-cell phospho-proteomics to map JAK2 activation heterogeneity

  • Mass cytometry (CyTOF) for comprehensive signaling network analysis

  • Imaging mass cytometry for spatial resolution of JAK2 activation in tissues

  • Spectral flow cytometry for multiplexed phospho-protein detection

• Combined genomic and phospho-protein analysis:

  • CITE-seq approaches incorporating phospho-JAK2 antibodies

  • Correlation of JAK2 mutations with phosphorylation status at single-cell level

  • Spatial transcriptomics combined with phospho-JAK2 imaging

• Technological advances:

  • Development of brighter fluorophore conjugates for improved sensitivity

  • Antibody engineering for enhanced cell permeability

  • Novel fixation methods preserving phospho-epitopes while enabling genomic analysis

These developments will provide unprecedented insights into cellular heterogeneity in JAK2-dependent signaling and disease processes .

What novel research questions can be addressed using phospho-JAK2 antibodies in combination with other molecular tools?

Integrating phospho-JAK2 antibodies with other research tools enables exploration of:

• Regulatory mechanisms of JAK2 signaling:

  • Combining CRISPR/Cas9 gene editing with phospho-JAK2 detection to identify novel regulators

  • Using proximity labeling techniques (BioID, APEX) to map phospho-JAK2 interaction networks

  • Implementing optogenetic control of JAK2 activation with phospho-antibody readouts

• Spatial-temporal dynamics:

  • Live-cell imaging using nanobody-based sensors derived from phospho-JAK2 antibodies

  • Super-resolution microscopy to visualize JAK2 signaling nanoclusters

  • Correlative light-electron microscopy for ultrastructural context of JAK2 activation

• Therapeutic development:

  • High-content screening using phospho-JAK2 antibodies to identify novel inhibitors

  • Patient-derived organoid testing with phospho-JAK2 readouts for personalized medicine

  • Development of JAK2-targeted degraders with phospho-JAK2 as pharmacodynamic marker