JAKMIP1 Antibody

What is JAKMIP1 and what are its primary functions?

JAKMIP1, also known as Marlin-1, is a highly-conserved vertebrate-specific microtubule-associated protein that plays dual roles in both the nervous and immune systems. Its primary functions include:

• Regulation of microtubule cytoskeleton rearrangements

• Involvement in JAK1 signaling pathways

• Role in microtubule-dependent transport of GABA-B receptors

• Function as an RNA binding protein in translational regulation

• Component of polyribosomes and RNP translational regulatory complexes

JAKMIP1 interacts mechanically with microtubules to regulate cytoskeletal dynamics, impacting processes such as cell shape and movement, highlighting its functional versatility .

What is the expression pattern of JAKMIP1 in different tissues?

JAKMIP1 exhibits a specific expression pattern that varies across tissues and isoforms:

• Predominantly expressed in neural tissues and lymphoid cells at the protein level

• Multiple isoforms show differential expression patterns:

  • Isoforms 2, 3, and 4 are specifically expressed in brain and retina

  • Isoforms 1 and 5 are also detected in liver, lung, and skeletal muscle

  • Expression also found in testis and to a lesser extent in spleen and intestine

This tissue-specific expression pattern provides important context when designing experiments using JAKMIP1 antibodies, as researchers should consider the most appropriate tissue controls based on the isoforms they aim to study.

How is JAKMIP1 implicated in autism spectrum disorders?

JAKMIP1 has emerged as a significant protein in autism spectrum disorder (ASD) research:

• Differentially expressed in patients with distinct syndromic forms of ASD, including Fragile X Syndrome and 15q duplication syndrome

• Eleven ASD subjects have been identified with copy number variations containing the JAKMIP1 gene

• Jakmip1 knockout in mouse models leads to ASD-related behaviors, including:

  • Motor and neurological stereotypies

  • Social abnormalities

  • Abnormal ultrasonic vocalizations

  • Reduced anxiety/increased impulsivity

  • Motor impairments

• JAKMIP1 loss dysregulates neuronal translation during synaptic development, affecting glutamatergic NMDAR signaling

• Strengthens the link between neuronal translation and behavior, an emerging theme in ASD pathophysiology

What criteria should be used when selecting a JAKMIP1 antibody for specific applications?

When selecting a JAKMIP1 antibody, researchers should consider the following criteria:

For more robust research outcomes, consider using two different antibodies targeting distinct epitopes of JAKMIP1 to confirm specificity of your findings.

How can I validate a JAKMIP1 antibody before using it in critical experiments?

Rigorous validation is essential before using JAKMIP1 antibodies in critical experiments:

• Positive control selection:

  • Use tissues known to express JAKMIP1, such as brain tissue (particularly cortex), spleen tissue, or cell lines like HEK-293, Jurkat, or Y79 cells

  • For western blot validation, rat brain whole cell lysate is commonly recommended

• Knockout/knockdown validation:

  • Ideally, validate using Jakmip1 knockout tissue or JAKMIP1 knockdown cells

  • Compare antibody signal between wildtype and knockout/knockdown samples

• Peptide competition assay:

  • Some antibodies (e.g., PA5-34495) have available blocking peptides (e.g., PEP-1537) that can be used to confirm specificity

  • Pre-incubate antibody with excess peptide antigen to block specific binding

• Multiple application testing:

  • Confirm antibody specificity across multiple applications (WB, IHC, IP)

  • Observe consistent molecular weight detection (~73 kDa) in western blots

• Isoform considerations:

  • If targeting specific isoforms, verify that the antibody detects the expected size variant

What are the optimal conditions for Western blot analysis using JAKMIP1 antibodies?

For optimal Western blot results with JAKMIP1 antibodies:

Sample preparation:

• Use fresh tissue or cells with high JAKMIP1 expression (brain, lymphoid tissues)

• Employ standard protein extraction methods with protease inhibitors

• For brain tissue, use established protocols to preserve protein integrity

Western blot parameters:

• Protein loading: 10-30 μg total protein per lane

• Expected molecular weight: 73 kDa

• Recommended antibody dilutions: Generally 1:500-1:2000, but specifically:

  • For PA5-34495: 1:1000

  • For 13846-1-AP: 1:500-1:1000

  • For DF12647: concentrations vary by application

  • For ABIN7115543: 1:500-1:2000

Detection example:
Western blot with rat brain whole cell lysate shows bands at the predicted size of 73 kDa when using 1-2 µg/mL of antibody ab211296 .

What protocol modifications are necessary for successful immunohistochemistry with JAKMIP1 antibodies?

For successful immunohistochemistry using JAKMIP1 antibodies:

Tissue preparation:

• Formalin-fixed paraffin-embedded (FFPE) or frozen sections are both compatible

• For FFPE sections of human brain cortex, demonstrated success has been shown with 10 μg/mL of ab211296

Antigen retrieval options:

• Primary recommendation: TE buffer pH 9.0

• Alternative: Citrate buffer pH 6.0

Antibody dilutions:

• For 13846-1-AP: 1:50-1:500

• For ABIN7115543: 1:20-1:200

Detection systems:

• Use appropriate secondary antibody detection systems based on host species (typically rabbit for available JAKMIP1 antibodies)

• Include appropriate positive controls (human brain cortex tissue) and negative controls

Special considerations:

• Higher background may occur in tissues with high endogenous biotin

• Consider using biotin-free detection systems if necessary

How can I optimize immunoprecipitation procedures using JAKMIP1 antibodies?

For effective immunoprecipitation with JAKMIP1 antibodies:

Antibody selection:

• Choose antibodies validated for IP applications:

  • 13846-1-AP has been validated for IP in Jurkat cells

  • ABIN7115543 recommends 1:200-1:1000 dilution for IP

Protocol parameters:

• Use 0.5-4.0 μg antibody per 1.0-3.0 mg of total protein lysate

• Pre-clear lysates to reduce non-specific binding

• Include appropriate negative controls (non-immune IgG)

Co-IP applications:
JAKMIP1 antibodies have been successfully used to confirm interactions with:

• DDX5, CLASP2, PABPC1, and CAMK2G in co-IP experiments

• FMRP in co-IP confirming JAKMIP1's presence in the FMRP-containing RNP complex

Verification methods:

• Validate successful IP by western blot analysis of immunoprecipitated material

• Confirm expected 73 kDa band specific to JAKMIP1

What are common issues with JAKMIP1 antibody detection and how can they be resolved?

When using JAKMIP1 antibodies in brain tissue, be aware that expression varies across regions and developmental stages, which may affect detection sensitivity.

How can I distinguish between JAKMIP1 isoforms using available antibodies?

Distinguishing between JAKMIP1 isoforms requires careful antibody selection and experimental design:

• Epitope mapping:

  • Review the immunogen sequence information provided with the antibody

  • PA5-41941 targets the peptide sequence: FLRLQVLEQQ HVIDDLSLER ERLLRSKRHR GKSLKPPKKH VVETFFGFDE

  • Determine if this sequence is present in all isoforms or is isoform-specific

• Molecular weight differentiation:

  • The canonical isoform has a predicted molecular weight of 73 kDa

  • Run gradient gels to better separate isoforms of similar sizes

  • Include positive controls for different tissues known to express specific isoforms:

    • Brain/retina tissue for isoforms 2-4

    • Liver/lung/muscle for isoforms 1 and 5

• RT-PCR complementation:

  • Use isoform-specific PCR primers alongside antibody detection

  • Correlate mRNA expression with protein detection to confirm isoform identity

• Isoform-specific antibodies:

  • Consider generating custom antibodies against unique regions of specific isoforms if commercially available antibodies cannot distinguish between them

How can JAKMIP1 antibodies be used to investigate its role in translational regulation?

JAKMIP1 antibodies can facilitate several approaches to study its role in translational regulation:

• Polyribosome profiling:

  • Use JAKMIP1 antibodies to detect its association with polyribosomes, monosomes, and mRNPs

  • Compare distribution patterns between wild-type and JAKMIP1 knockout/knockdown samples

  • Research has shown that JAKMIP1 loss causes a shift of PABPC1 and DDX5 proteins from polyribosome fractions to monosome and mRNP fractions

• Co-immunoprecipitation of translational complexes:

  • Use JAKMIP1 antibodies to pull down associated translational machinery components

  • Analyze the composition of JAKMIP1-associated RNP complexes

  • Previous studies have demonstrated that JAKMIP1 interacts with translational regulators including FMRP, DDX5, and PABPC1

• RNA-immunoprecipitation (RIP):

  • Employ JAKMIP1 antibodies to identify bound RNA targets

  • Characterize mRNAs regulated by JAKMIP1 in different neuronal populations

  • This approach can help identify the specific transcripts regulated by JAKMIP1 during development

• Proximity ligation assays:

  • Use JAKMIP1 antibodies in combination with antibodies against translational machinery components

  • Visualize direct interactions in situ within neuronal compartments

What experimental approaches can reveal JAKMIP1's contribution to autism-related mechanisms?

JAKMIP1 antibodies enable multiple approaches to investigate its role in autism-related mechanisms:

• Comparative protein expression analysis:

  • Compare JAKMIP1 expression levels in post-mortem brain tissue from ASD patients versus controls

  • Analyze regional and cellular distribution patterns using immunohistochemistry

  • Studies have already shown differential expression in Fragile X and 15q duplication syndromes

• Developmental expression profiling:

  • Track JAKMIP1 expression throughout brain development

  • Focus on critical periods of synaptogenesis and circuit formation

  • JAKMIP1 has been shown to play a role during the peak period of cortical synaptogenesis

• Glutamatergic signaling studies:

  • Use JAKMIP1 antibodies alongside NMDAR component antibodies

  • Investigate how JAKMIP1 knockout affects localization and expression of glutamate receptors

  • Research has shown that JAKMIP1 loss leads to glutamatergic NMDAR signaling deficits

• Interaction with FMRP pathway components:

  • Employ co-immunoprecipitation to characterize JAKMIP1's relationship with FMRP

  • Compare binding partners between wild-type and autism model systems

  • JAKMIP1 has been confirmed as part of the FMRP-kinesin transport RNP granule

• Synaptic protein translation analysis:

  • Use JAKMIP1 antibodies to study local translation at synapses

  • Investigate how JAKMIP1 deficiency affects activity-dependent protein synthesis

  • This can reveal mechanisms connecting translational dysregulation to autism phenotypes

How can JAKMIP1 antibodies facilitate investigation of its dual roles in nervous and immune systems?

JAKMIP1 functions in both neural and immune contexts, and antibodies can help elucidate these dual roles:

• Comparative tissue analysis:

  • Use immunohistochemistry to compare JAKMIP1 expression patterns between:

    • Neural tissues (brain regions, primary neurons)

    • Immune tissues (lymph nodes, spleen)

    • Mixed neural-immune interfaces (microglia, neuroimmune junctions)

• Cell-type specific localization:

  • Apply immunofluorescence with cell-type markers to determine:

    • Neuronal vs. glial expression patterns

    • Distribution across lymphocyte populations

    • Subcellular localization differences between cell types

• Cytoskeletal interaction studies:

  • Use co-immunoprecipitation to compare JAKMIP1's binding partners between:

    • Neuronal microtubule complexes

    • Immune cell cytoskeletal components

  • JAKMIP1 mechanically interacts with microtubules and regulates cytoskeletal dynamics

• JAK1 signaling pathway analysis:

  • Investigate how JAKMIP1 regulates JAK1 signaling differently in:

    • Neurons during development

    • Immune cells during activation

  • This could reveal tissue-specific regulatory mechanisms

• Polarized secretion in lymphocytes:

  • Study JAKMIP1's role in T cell cytotoxicity using antibodies to track:

    • Protein localization during immune synapse formation

    • Association with secretory machinery

  • Research suggests JAKMIP1 participates in polarized secretion in lymphocytes and may restrain T cell-mediated cytotoxicity

What are the optimal conditions for multiplexed immunofluorescence including JAKMIP1?

For successful multiplexed immunofluorescence with JAKMIP1 antibodies:

• Primary antibody combination strategy:

  • Use JAKMIP1 rabbit polyclonal antibodies with mouse monoclonals against other targets

  • Avoid antibodies raised in the same species unless using specialized methods

  • Test each antibody individually before combining

• Signal optimization:

  • Titrate JAKMIP1 antibody concentration (typically 1:50-1:500 range for IF)

  • Optimize antigen retrieval methods for all targets simultaneously

  • Consider sequential immunostaining for difficult combinations

• Co-localization studies:

  • Pair JAKMIP1 antibodies with markers for:

    • Microtubules (to study cytoskeletal interactions)

    • GABA-B receptors (to examine transport roles)

    • Polyribosomes (to investigate translational functions)

    • FMRP complexes (to study autism-related mechanisms)

• Controls and validation:

  • Include single-stained controls for spectral overlap correction

  • Use knockout/knockdown tissues as negative controls

  • Consider additional blocking steps to minimize cross-reactivity

When examining neuronal tissues, note that JAKMIP1 shows high expression in glutamatergic neurons during brain development, which should inform experimental design and interpretation.

What considerations are important when using JAKMIP1 antibodies in primary neuronal cultures?

When working with primary neuronal cultures:

• Developmental timing:

  • JAKMIP1 expression varies throughout neuronal development

  • For synaptic studies, cultures should be maintained until proper synaptic development (14-21 DIV)

  • JAKMIP1 has significant roles during peak periods of cortical synaptogenesis

• Fixation and permeabilization:

  • For optimal preservation of JAKMIP1's microtubule association:

    • Use 4% paraformaldehyde fixation (10-15 minutes)

    • Mild permeabilization with 0.1-0.2% Triton X-100

    • Consider methanol fixation for certain applications

• Cell-type considerations:

  • JAKMIP1 is highly expressed in glutamatergic neurons

  • Use appropriate neuronal subtype markers for co-localization studies

  • Consider the composition of your culture system when interpreting results

• Subcellular localization studies:

  • JAKMIP1 associates with microtubules and polyribosomes

  • Use high-resolution imaging to examine distribution in:

    • Dendrites and dendritic spines

    • Growth cones during development

    • RNA granules and local translation sites

• Activity-dependent changes:

  • Investigate how neuronal activity affects JAKMIP1 localization

  • Consider treatments that modulate translation or cytoskeletal dynamics