JAKMIP2 Antibody

Biological Context and Expression of JAKMIP2

JAKMIP2, also known as neuroendocrine long coiled-coil protein 1 (NECC1), CTCL tumor antigen HD-CL-04, or JAMIP2, is an 810 amino acid protein belonging to the JAKMIP family . This protein primarily localizes to the Golgi apparatus and demonstrates high expression in brain tissue, with moderate expression levels in thymus, spleen, and lung . The protein exists in three alternatively spliced isoforms, with the gene encoding JAKMIP2 mapping to human chromosome 5q32 and mouse chromosome 18 B3 . The molecular weight of JAKMIP2 is approximately 95 kDa, which aligns with both its calculated and observed weights in experimental contexts .

Available JAKMIP2 Antibody Types and Specifications

Multiple manufacturers produce polyclonal antibodies against JAKMIP2, primarily raised in rabbits . These antibodies display reactivity with human, mouse, and rat samples, making them versatile tools for comparative studies across species . The antibodies are typically supplied in liquid form, purified through antigen affinity methods, and stored in PBS buffer containing sodium azide and glycerol at pH 7.3-7.4 . Most commercial preparations have been validated for Western blot applications, with some also tested for ELISA, immunohistochemistry (IHC), and immunocytochemistry/immunofluorescence (ICC/IF) .

Proper Storage and Handling of JAKMIP2 Antibodies

For optimal results with JAKMIP2 antibodies, storage at -20°C is universally recommended by manufacturers . These antibodies typically remain stable for one year after shipment when stored appropriately . While some preparations indicate that aliquoting is unnecessary for -20°C storage, it is generally good practice to create single-use aliquots to prevent freeze-thaw cycles that can degrade antibody quality and specificity . Several commercial preparations are formulated with 50% glycerol, which helps maintain antibody stability during freeze-thaw cycles when necessary .

Optimizing Western Blot Protocols for JAKMIP2 Detection

For Western blot applications, JAKMIP2 antibodies should be employed at dilutions ranging from 1:500 to 1:3000, with most manufacturers recommending the 1:1000-1:3000 range for optimal results . Given the 95 kDa size of JAKMIP2, standard polyacrylamide gel concentrations of 8-10% are appropriate for adequate protein separation . When performing Western blot analysis of JAKMIP2, brain tissue samples from human, mouse, and rat have consistently demonstrated positive detection . It is advisable to include positive control lysates from brain tissue when validating new antibody lots or experimental conditions .

Immunohistochemistry and Immunocytochemistry Considerations

For immunohistochemistry applications, JAKMIP2 antibodies should be used at more concentrated dilutions, typically 1:50-1:100 . This higher concentration compensates for the more complex tissue environment and potential epitope masking that occurs during fixation processes. When conducting immunocytochemistry experiments, researchers should consider the subcellular localization of JAKMIP2 to the Golgi apparatus when interpreting results . Co-staining with established Golgi markers can provide valuable validation of antibody specificity and proper localization patterns .

Cross-Reactivity and Species Compatibility Analysis

The available JAKMIP2 antibodies demonstrate confirmed reactivity with human, mouse, and rat samples . This cross-species reactivity is supported by the high sequence conservation of JAKMIP2 across these species, with sequence identity reaching 100% with mouse and 99% with rat for certain immunogenic regions . This high conservation facilitates translational research between rodent models and human samples. Researchers should note that while these antibodies have been validated for the three species mentioned, testing in other species would require validation by the end user .

Investigating JAKMIP2 Isoforms and Splice Variants

JAKMIP2 exists in three alternatively spliced isoforms, which presents both challenges and opportunities for research . When investigating specific isoforms, researchers should carefully select antibodies with known epitope locations relative to splicing regions. The JAKMIP2 antibody from Thermo Fisher (PA5-66740) is raised against an immunogen sequence "RETEKQCKPLL ERNKCLAKRN DELMVSLQRM EEKLKAVTKE NSEMREKITS HPPLKKLKSL NDLDQANEEQ," which may be useful for distinguishing certain isoforms depending on the sequence conservation across splice variants . For definitive isoform discrimination, complementary techniques such as RT-PCR with isoform-specific primers should be employed alongside antibody-based detection methods.

Antibody Validation Strategies for JAKMIP2 Research

Rigorous validation of JAKMIP2 antibodies is critical for ensuring experimental reliability. Multiple orthogonal approaches should be employed, including:

• Positive control tissues with known JAKMIP2 expression (brain tissue is strongly recommended)

• Negative controls using tissues with minimal expression or knockout/knockdown samples

• Peptide competition assays using the immunizing peptide (when available from the manufacturer)

• Comparison of results across multiple antibodies targeting different epitopes of JAKMIP2

• Correlation of protein detection with mRNA expression data from the same samples

These validation steps are particularly important when studying JAKMIP2 in novel contexts or disease models where expression patterns may differ from established norms.

Troubleshooting Weak or Non-specific JAKMIP2 Signal

When encountering weak or non-specific signals with JAKMIP2 antibodies, several protocol modifications should be considered:

• For Western blotting:

  • Adjust antibody concentration within the recommended range (1:500-1:3000)

  • Optimize blocking conditions to reduce background (5% non-fat milk or BSA)

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

  • Ensure adequate protein loading (50-100 μg total protein for cell/tissue lysates)

  • Test different membrane types (PVDF vs. nitrocellulose)

• For immunohistochemistry:

  • Test different antigen retrieval methods (heat-induced vs. enzymatic)

  • Increase antibody concentration (toward the 1:50 dilution range)

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

  • Test different detection systems (HRP/DAB vs. fluorescent secondary antibodies)

• For immunocytochemistry:

  • Compare different fixation methods (paraformaldehyde vs. methanol)

  • Optimize permeabilization conditions (0.1-0.5% Triton X-100)

  • Include a blocking step with normal serum from the secondary antibody host species

JAKMIP2 in Neurological Research Models

Given the high expression of JAKMIP2 in brain tissue , neurological research represents a primary application area for JAKMIP2 antibodies. When designing experiments in this field, researchers should consider:

• Brain region-specific expression patterns of JAKMIP2

• Potential co-localization with neuronal or glial markers

• Changes in JAKMIP2 expression during development or in pathological conditions

• Interaction with microtubules and implications for neuronal transport mechanisms

The antibody's demonstrated reactivity with human, mouse, and rat brain tissues facilitates comparative studies and translation between model organisms and human samples .

Investigating JAKMIP2 Interactions with Janus Kinase Pathways

As suggested by its name (Janus kinase and microtubule interacting protein), JAKMIP2 may play roles in JAK signaling pathways . When exploring these potential interactions, researchers should consider:

• Co-immunoprecipitation experiments to identify JAKMIP2-JAK protein complexes

• Analysis of phosphorylation states in response to cytokine stimulation

• Localization changes following pathway activation

• Functional consequences of disrupting JAKMIP2-JAK interactions

For such studies, careful controls and validation steps are essential to distinguish specific from non-specific interactions.

JAKMIP2 in Chromosome 5q32-related Pathologies

JAKMIP2 maps to human chromosome 5q32, a region implicated in various pathological conditions . Deletion of chromosome 5q is associated with therapy-related acute myelogenous leukemias and myelodysplastic syndrome . Additionally, chromosome 5 contains approximately 6% of the human genome, with deletions of the p arm linked to Cri du chat syndrome . When investigating JAKMIP2's potential involvement in these conditions, researchers should:

• Compare JAKMIP2 expression in normal vs. pathological samples

• Analyze correlation between JAKMIP2 levels and disease progression

• Consider potential functional consequences of altered JAKMIP2 expression

• Investigate whether JAKMIP2 serves as a biomarker for specific conditions

Appropriate Controls for JAKMIP2 Antibody Experiments

Rigorous experimental design for JAKMIP2 research should include the following controls:

• Positive tissue controls: Brain tissue is strongly recommended, with thymus, spleen, and lung as alternative positive controls

• Negative controls: Tissues with minimal JAKMIP2 expression or isotype control antibodies

• Technical controls: Secondary antibody-only controls to assess non-specific binding

• Validation controls: Peptide competition assays when applicable

These controls help distinguish specific from non-specific signals and provide confidence in experimental outcomes.

Quantitative Analysis of JAKMIP2 Expression

For quantitative analysis of JAKMIP2 expression:

• Western blot: Include standard curves with recombinant protein or well-characterized samples

• Immunohistochemistry: Employ digital image analysis with appropriate normalization

• Flow cytometry: Use calibration beads to standardize fluorescence intensity

Careful selection of housekeeping genes or proteins for normalization is essential, particularly when comparing across tissues or disease states.