JAK2 (Ab-570) Antibody

What is JAK2 (Ab-570) Antibody and what epitope does it recognize?

JAK2 (Ab-570) Antibody is a rabbit polyclonal antibody that detects endogenous levels of total JAK2 protein. It specifically recognizes the peptide sequence around amino acids 568-572 (G-D-Y-G-Q) derived from human JAK2 . The antibody is produced by immunizing rabbits with a synthetic peptide conjugated to KLH (Keyhole Limpet Hemocyanin), followed by affinity purification using epitope-specific peptide . This antibody targets a region containing tyrosine 570, which is a significant regulatory phosphorylation site in JAK2 .

What applications has JAK2 (Ab-570) Antibody been validated for?

The antibody has been validated for several research applications:

• Immunohistochemistry (IHC): Typically used at dilutions of 1:50-1:100

• Western Blotting (WB): Optimal dilution range of 1:500-1:3000

• Immunofluorescence (IF): Recommended dilution of 1:100-1:500

Scientific validation data includes successful detection of JAK2 in human breast carcinoma tissue by IHC and in 293 cells treated with etoposide (25μM, 24 hours) by Western blot . The antibody enables researchers to examine JAK2 expression patterns and study the functional significance of the Y570 region in various experimental contexts.

What species reactivity does JAK2 (Ab-570) Antibody demonstrate?

Different manufacturers may offer slightly different reactivity profiles for their JAK2 (Ab-570) antibodies:

The antibody has been consistently validated for human JAK2, with varying cross-reactivity to rodent JAK2 proteins . When using this antibody with species not explicitly listed in the manufacturer's specifications, preliminary validation is strongly recommended.

What is the significance of tyrosine 570 in JAK2 function?

Tyrosine 570 (Y570) is a critical regulatory site within JAK2's pseudokinase domain (JH2):

• Y570 is a prominent site of autophosphorylation in JAK2

• Phosphorylation of Y570 has an inhibitory effect on JAK2 kinase activity

• It contributes to rapid termination of ligand-activated JAK2 signaling

• Y570 phosphorylation helps maintain low basal JAK2 activity in unstimulated cells

• Mutation of Y570 to phenylalanine (Y570F) increases JAK2 activation and downstream STAT phosphorylation

In the structural context, phosphorylated Y570 appears to stabilize an autoinhibitory interaction between the pseudokinase (JH2) and kinase (JH1) domains of JAK2. Molecular dynamics simulations suggest that phosphorylated Y570 can insert into a pocket formed by the β-sheet in the N-lobe of JH1, forming salt bridges with Lys883, Lys926, and Arg922 . This interaction helps maintain JAK2 in an inactive conformation until appropriate stimulation occurs.

How does the pseudokinase domain (JH2) regulate JAK2 through Y570 phosphorylation?

Contrary to the traditional view of JH2 as a catalytically inactive pseudokinase domain, research has demonstrated that JAK2's JH2 domain functions as a dual-specificity protein kinase capable of phosphorylating both Ser523 and Tyr570 . This creates an intricate autoregulatory mechanism:

• JH2 catalyzes the phosphorylation of Ser523 first

• Phosphorylation of Ser523 is required for subsequent phosphorylation of Tyr570

• The K581A mutation in JH2, which disrupts ATP binding, abolishes phosphorylation of both Ser523 and Tyr570

• This phosphorylation occurs independently of the kinase activity of JH1, as demonstrated by studies using JAK2 constructs with either inactivated JH1 (K882D) or complete deletion of JH1

This sequential phosphorylation mechanism creates a regulatory circuit where JH2 self-limits JAK2 activity through Y570 phosphorylation. The JH2 domain appears to have evolved as an integrated regulatory module that maintains JAK2 in an appropriate activation state through this auto-phosphorylation capacity .

What experimental evidence supports the regulatory role of Y570 phosphorylation?

Multiple experimental approaches have established Y570 as a negative regulatory site:

• Mutational Analysis:

  • Y570F mutation (preventing phosphorylation) increases JAK2 tyrosine phosphorylation at the JH1 activation loop (Tyr1007-Tyr1008)

  • Y570F mutants show enhanced STAT phosphorylation and signaling compared to wild-type JAK2

  • Charge-reversal mutation Y570R activates JAK2 approximately 4-fold relative to wild-type

• Domain-Specific Studies:

  • JAK2 constructs lacking the entire JH1 domain still exhibit phosphorylation of Ser523 and Tyr570, confirming JH2's catalytic role

  • K581A mutation in JH2 (disrupting ATP binding) abolishes phosphorylation of both Ser523 and Tyr570

• Structural Investigations:

  • Molecular dynamics simulations reveal that pY570 can form salt bridges with Lys883, Lys926, and Arg922 in the JH1 domain

  • The double mutant Y570R K883E shows suppressed activation, consistent with formation of a "reverse" salt bridge that restores autoinhibition

• Cytokine Signaling Impact:

  • JAK2 mutants S523A, Y570F, and K581A all show increased basal phosphorylation of STAT1

  • These mutations affect cytokine-induced transcriptional responses, highlighting the physiological relevance of Y570 phosphorylation

How can JAK2 (Ab-570) Antibody be used to study JAK2 phosphorylation dynamics?

JAK2 (Ab-570) Antibody serves as a valuable tool for investigating JAK2 regulation, but it's important to understand its capabilities and limitations:

• Detection of Total JAK2: The antibody recognizes total JAK2 protein regardless of Y570 phosphorylation status

• Requires Complementary Antibodies: For comprehensive analysis of Y570 phosphorylation:

  • Use JAK2 (Ab-570) Antibody to detect total JAK2 levels

  • Use phospho-specific antibodies (e.g., α-pY570 JAK2) to detect phosphorylated Y570

  • Include antibodies against other phosphorylation sites (pY1007/1008, pY221) to assess activation state

Experimental Approach for Phosphorylation Studies:

• Stimulation Time Course:

  • Treat cells with relevant cytokines (e.g., EPO, IL-6, IFN-γ)

  • Collect samples at multiple time points

  • Analyze with JAK2 (Ab-570) Antibody and phospho-specific antibodies

  • Correlate Y570 phosphorylation with JAK2 activation and downstream signaling

• Phosphatase Treatment Controls:

  • Split samples and treat one set with phosphatases

  • Compare phospho-specific antibody binding before and after treatment

  • JAK2 (Ab-570) Antibody signal should remain unchanged

• Mutational Validation:

  • Express wild-type JAK2 and Y570F mutant

  • Confirm similar detection with JAK2 (Ab-570) Antibody

  • Verify absence of signal with phospho-Y570 antibody in the mutant

What is the relationship between Y570 phosphorylation and JAK2-associated diseases?

The regulatory role of Y570 phosphorylation has important implications for JAK2-associated pathologies:

• Myeloproliferative Neoplasms (MPNs):

  • The JAK2 V617F mutation causes constitutive JAK2 activation in a high percentage of MPN patients

  • V617F and other MPN-associated mutations in the JH2 domain have been found to abrogate JH2 activity

  • This would reduce phosphorylation of inhibitory sites like Y570, contributing to pathological JAK2 hyperactivation

• Acute Lymphoblastic Leukemia (ALL):

  • Mutations like R683S in JH2 and D873N in JH1 affect the JH2-JH1 interface near the region where pY570 interacts

  • These mutations likely disrupt the autoinhibitory mechanism involving Y570, leading to inappropriate JAK2 activation

• Research Applications:

  • JAK2 (Ab-570) Antibody can be used alongside phospho-specific antibodies to assess Y570 phosphorylation status in patient samples

  • Comparing the ratio of phosphorylated Y570 to total JAK2 between healthy and diseased samples may provide diagnostic insights

  • The antibody facilitates research on compounds that might restore normal JAK2 regulation in disease states

What technical considerations are important for optimizing JAK2 (Ab-570) Antibody use?

Successful application of JAK2 (Ab-570) Antibody requires attention to several technique-specific factors:

Immunohistochemistry (IHC) Optimization:

• Recommended dilution: 1:50-1:100

• Validated positive control: Human breast carcinoma tissue

• Fixation: Compatible with formalin-fixed, paraffin-embedded tissues

• Antigen retrieval: May be necessary to expose the epitope

• Detection: Works with standard visualization systems (DAB, AEC)

Western Blotting (WB) Protocol Refinement:

• Recommended dilution: 1:500-1:3000

• Expected molecular weight: ~131 kDa for full-length JAK2

• Sample preparation: Include phosphatase inhibitors to preserve phosphorylation

• Blocking: 5% BSA in TBST is often preferable to milk for phospho-proteins

• Positive control: 293 cells treated with etoposide (25μM, 24 hours)

Immunofluorescence (IF) Considerations:

• Recommended dilution: 1:100-1:500

• Fixation: 4% paraformaldehyde followed by permeabilization

• Counterstain: DAPI for nuclear visualization

• Controls: Include secondary-only control to assess background

Storage and Handling:

• Store at -20°C for long-term preservation

• Can be stored at 4°C for short-term use

• Formulation: Supplied at 1.0mg/mL in phosphate buffered saline (without Mg2+ and Ca2+), pH 7.4, containing 150mM NaCl, 0.02% sodium azide, and 50% glycerol

• Avoid repeated freeze-thaw cycles to maintain antibody performance