JAK2 (Ab-1007) Antibody

What is JAK2 (Ab-1007) Antibody and what epitope does it recognize?

JAK2 (Ab-1007) Antibody is a rabbit polyclonal antibody that recognizes a specific peptide sequence around amino acids 1005-1009 (K-E-Y-Y-K) derived from Human JAK2. This antibody is designed to detect endogenous levels of total JAK2 protein, not just phosphorylated forms . It is supplied at a concentration of 1.0mg/mL in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol .

What applications is JAK2 (Ab-1007) Antibody validated for?

The JAK2 (Ab-1007) Antibody has been validated for Western Blot (WB) and ELISA applications . For Western blotting, the recommended dilution range is 1:500-1:1000 . Validation studies have demonstrated successful detection of JAK2 in extracts from multiple cell lines including HeLa, 3T6, and 293 cells .

What species reactivity does JAK2 (Ab-1007) Antibody exhibit?

JAK2 (Ab-1007) Antibody shows cross-reactivity with human, mouse, and rat species . This multi-species reactivity makes it valuable for comparative studies across different model organisms. Researchers should note that the predicted molecular weight of the detected JAK2 protein is approximately 125 kDa .

How should JAK2 (Ab-1007) Antibody be stored for optimal stability?

For long-term preservation, it is recommended to store JAK2 (Ab-1007) Antibody at -20°C or -80°C . For short-term use (within a few weeks), storage at 4°C is acceptable. It's important to avoid repeated freeze-thaw cycles as they may compromise antibody performance . Proper aliquoting upon receipt is recommended for antibodies that will be used multiple times over an extended period.

How can JAK2 (Ab-1007) Antibody be used to study JAK2 phosphorylation states in signal transduction research?

While JAK2 (Ab-1007) Antibody detects total JAK2 protein regardless of phosphorylation state, it can be used in conjunction with phospho-specific antibodies (such as those targeting phospho-Y1007/Y1008) to investigate JAK2 activation . A recommended experimental approach includes:

• Performing parallel Western blots with both JAK2 (Ab-1007) and phospho-specific JAK2 antibodies

• Calculating the ratio of phosphorylated to total JAK2 to quantify activation status

• Including appropriate stimulation controls (e.g., cytokine treatment) and inhibitor treatments

This dual-antibody approach allows researchers to normalize phosphorylation signals to total protein levels, providing more accurate quantification of JAK2 activation in response to experimental treatments .

What controls should be included when using JAK2 (Ab-1007) Antibody in Western blot experiments?

For rigorous Western blot experiments with JAK2 (Ab-1007) Antibody, the following controls should be included:

Including these controls helps ensure the reliability and interpretability of results, particularly in complex signaling studies where multiple proteins and phosphorylation events may be analyzed simultaneously .

How can JAK2 (Ab-1007) Antibody be used to investigate JAK2 variants such as the 45-kDa truncated form?

The recently identified 45-kDa JAK2 variant (FERM-JAK2) lacks a major part of the FERM domain, the entire SH2-like domain, and the pseudokinase domain, resulting in a fusion protein consisting of the N-terminal 77 amino acids together with residues 814-1132 of the kinase domain . To study this variant:

• Use JAK2 (Ab-1007) Antibody which recognizes the C-terminal region (aa. 1005-1009) present in both full-length and truncated variants

• Run SDS-PAGE gels with conditions optimized to resolve proteins in both the 125 kDa and 45 kDa range

• Compare with antibodies targeting different JAK2 epitopes (e.g., N-terminal or pseudokinase domain) to confirm variant identity

• Consider immunoprecipitation followed by mass spectrometry for definitive identification

This approach has been used successfully to identify the FERM-JAK2 variant in ruxolitinib-resistant cell clones, demonstrating persistent activation of STAT5 despite JAK2 inhibitor treatment .

What are the most common causes of non-specific bands when using JAK2 (Ab-1007) Antibody in Western blot, and how can they be minimized?

Non-specific bands are a common challenge in Western blot experiments. With JAK2 (Ab-1007) Antibody, potential causes and solutions include:

• Cross-reactivity with related kinases:

  • Increase antibody dilution (try 1:1000 instead of 1:500)

  • Optimize blocking conditions (5% BSA may be preferable to milk for phospho-proteins)

  • Consider using more stringent washing buffers

• Detection of JAK2 degradation products:

  • Add fresh protease inhibitors to lysis buffer

  • Keep samples cold throughout preparation

  • Reduce sample processing time

• Alternative splice variants:

  • Use additional JAK2 antibodies targeting different epitopes to confirm identity

  • Compare with known JAK2 variant expression patterns

• Non-specific binding:

  • Increase blocking time (overnight at 4°C may help)

  • Try alternative blocking agents (casein, commercial blockers)

  • Include 0.1-0.5% Tween-20 in antibody dilution buffer

Careful optimization of these parameters can significantly improve specificity and reduce background in Western blot experiments .

How can JAK2 (Ab-1007) Antibody be incorporated into kinase activity assays?

While JAK2 (Ab-1007) Antibody is not directly used in kinase activity measurements, it can play a valuable role in these experiments:

• Immunoprecipitation-based kinase assays:

  • Use JAK2 (Ab-1007) Antibody to immunoprecipitate JAK2 from cell lysates

  • Subject immunoprecipitates to in vitro kinase reactions with appropriate substrates (e.g., GST-JAK2CT containing Y1007/Y1008 sites)

  • Detect phosphorylation by autoradiography or phospho-specific antibodies

• Validation of active JAK2 levels:

  • Perform Western blot with JAK2 (Ab-1007) Antibody on input samples for kinase assays

  • Normalize kinase activity measurements to total JAK2 protein levels

  • Compare with phospho-Y1007 antibody results to correlate activity with activation status

This combined approach allows researchers to link kinase activity measurements with protein expression levels, providing more complete characterization of JAK2 function in experimental systems .

How can JAK2 (Ab-1007) Antibody be used to investigate the effects of JAK2 mutations in disease models?

JAK2 mutations, particularly V617F, play important roles in myeloproliferative neoplasms and other diseases. To study these mutations:

• Comparative analysis of mutant vs. wild-type JAK2:

  • Express wild-type JAK2 and JAK2-V617F in cell models

  • Perform Western blot with JAK2 (Ab-1007) Antibody to confirm equal expression

  • Compare downstream signaling activation (STAT5, ERK, AKT) using phospho-specific antibodies

• Patient sample analysis:

  • Extract proteins from patient samples carrying JAK2 mutations

  • Use JAK2 (Ab-1007) Antibody to assess total JAK2 levels

  • Compare with phospho-JAK2 levels to determine activation status

• Inhibitor studies:

  • Treat JAK2 mutant models with selective inhibitors (e.g., ruxolitinib)

  • Monitor both total JAK2 (using Ab-1007) and phospho-JAK2 levels

  • Assess resistance mechanisms, including potential expression of JAK2 variants

These approaches have revealed important insights, such as the role of the 45-kDa JAK2 variant in drug resistance and the differential effects of mutations on JAK2 activity and substrate preference .

What methodological considerations are important when using JAK2 (Ab-1007) Antibody to study JAK-STAT pathway activation in different cellular contexts?

The JAK-STAT pathway functions differently across cell types and stimulation conditions. When using JAK2 (Ab-1007) Antibody in these studies:

• Cell-type specific considerations:

  • Different cell types express varying levels of JAK2 and pathway components

  • Adjust antibody dilutions based on expression levels (higher dilutions for high-expressing cells)

  • Include appropriate positive control cell lines (e.g., HeLa, 3T6) alongside experimental samples

• Stimulation protocols:

  • Optimize cytokine concentration and stimulation time (e.g., EPO at 40 U/ml)

  • Include time course analysis to capture transient phosphorylation events

  • Consider pre-treatment with phosphatase inhibitors to preserve phosphorylation

• Co-receptor analysis:

  • JAK2 functions with multiple receptors (EPOR, CRLF2, etc.)

  • Consider co-immunoprecipitation studies to examine JAK2-receptor interactions

  • Compare receptor phosphorylation status with JAK2 activation

• Subcellular localization:

  • Consider subcellular fractionation to examine nuclear vs. cytoplasmic JAK2

  • Use JAK2 (Ab-1007) Antibody in immunofluorescence studies (after validation)

  • Correlate localization with activation state and function

These methodological considerations help ensure robust and physiologically relevant results when studying this complex signaling pathway across different experimental systems .

How can JAK2 (Ab-1007) Antibody be used in conjunction with selective JAK inhibitors to investigate resistance mechanisms in cancer models?

JAK inhibitor resistance is an emerging clinical challenge. To investigate resistance mechanisms:

• Development of resistant cell models:

  • Generate resistant cell lines through long-term exposure to increasing inhibitor concentrations

  • Monitor JAK2 expression using JAK2 (Ab-1007) Antibody during resistance development

  • Screen for emergence of JAK2 variants or altered expression patterns

• Comparative signaling analysis:

  • Assess pathway activation downstream of JAK2 in sensitive vs. resistant cells

  • Determine if JAK2 phosphorylation status correlates with inhibitor sensitivity

  • Investigate alternative signaling pathways that may compensate for JAK2 inhibition

• Combination therapy assessment:

  • Test JAK2 inhibitors in combination with inhibitors of alternative pathways

  • Use JAK2 (Ab-1007) Antibody to confirm target engagement

  • Monitor for changes in JAK2 expression or localization during treatment

Research using these approaches has identified important resistance mechanisms, including the expression of the truncated 45-kDa JAK2 variant that contains the kinase domain but lacks the inhibitor-binding pseudokinase domain, resulting in persistent STAT5 activation despite JAK inhibitor treatment .

How should researchers interpret differences in JAK2 detection patterns when comparing results from antibodies targeting different JAK2 epitopes?

Different JAK2 antibodies may produce varying detection patterns due to:

• Epitope accessibility:

  • JAK2 (Ab-1007) targets amino acids 1005-1009 in the kinase domain

  • This region may be differently accessible depending on JAK2 activation state

  • Compare with antibodies targeting other domains (FERM, pseudokinase) for complete characterization

• Protein variants:

  • JAK2 (Ab-1007) will detect truncated variants that retain the kinase domain

  • N-terminal targeting antibodies will miss C-terminal fragments

  • Use multiple antibodies to map which domains are present in observed bands

• Post-translational modifications:

  • Some modifications may mask epitopes

  • Compare native vs. denatured/reduced samples

  • Consider using phosphatase treatment to determine if modifications affect detection

The use of complementary antibodies targeting different epitopes has been crucial in identifying the 45-kDa JAK2 variant, which is detected by C-terminal antibodies but not by antibodies recognizing the pseudokinase domain .

What are the critical technical parameters for optimizing immunoprecipitation procedures using JAK2 (Ab-1007) Antibody?

For successful JAK2 immunoprecipitation:

• Lysis buffer composition:

  • Use buffers containing 50 mM β-glycerophosphate (pH 7.3), 5 mM EDTA, 1 mM EGTA, 5 mM β-mercaptoethanol, 1% Triton X-100, 0.1 M NaCl, and protease inhibitor cocktail

  • Add phosphatase inhibitors (sodium orthovanadate, sodium fluoride) to preserve phosphorylation

• Antibody coupling:

  • Use 2-5 μg of JAK2 (Ab-1007) Antibody per mg of total protein

  • Consider pre-coupling to Protein A/G beads for cleaner results

  • Include appropriate IgG controls to identify non-specific binding

• Incubation conditions:

  • Overnight incubation at 4°C generally yields better results than shorter incubations

  • Use gentle rotation rather than shaking to preserve protein-antibody interactions

  • Include 0.1-0.2% BSA in binding buffer to reduce non-specific binding

• Washing stringency:

  • Balance between stringency (to reduce background) and maintaining specific interactions

  • Consider a gradual decrease in detergent concentration during sequential washes

  • Perform final wash in detergent-free buffer if subsequent kinase assays are planned

These optimized conditions have been successfully used for JAK2 immunoprecipitation in studies of JAK2 kinase activity and protein-protein interactions .