MAP3K10 Antibody

What is MAP3K10 and what are its key biological functions?

MAP3K10 belongs to the protein kinase superfamily and functions as a mitogen-activated protein kinase kinase kinase. It participates in cellular signaling pathways, particularly the JUN N-terminal pathway activation . This kinase influences cellular dynamics through its role in signal transduction cascades and functions as a component of signaling complexes . MAP3K10 has a calculated molecular weight of approximately 103.7 kDa, though it is commonly observed at 72 kDa in experimental contexts .

What applications are MAP3K10 antibodies validated for?

MAP3K10 antibodies have been validated for multiple research applications, including Western blotting (WB), immunohistochemistry (IHC), immunohistochemistry with paraffin-embedded sections (IHC-p), immunocytochemistry (ICC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) . Different antibodies may be optimized for specific applications, so researchers should select antibodies validated for their intended experimental approach.

What species reactivity can be expected from available MAP3K10 antibodies?

Most commercially available MAP3K10 antibodies demonstrate reactivity to human and mouse samples . Some antibodies show broader cross-reactivity with additional species such as rat, dog, rabbit, and monkey . When selecting an antibody for non-human or non-mouse species, researchers should verify sequence homology between their target species and the immunogen sequence used to generate the antibody .

What are the recommended storage conditions for MAP3K10 antibodies?

For long-term storage, MAP3K10 antibodies should be stored at -20°C for up to one year . For frequent use and short-term storage (up to one month), 4°C is appropriate . It is advisable to avoid repeated freeze-thaw cycles as this can lead to antibody degradation and reduced performance in experimental applications .

How should researchers determine the appropriate dilution for MAP3K10 antibodies?

Recommended dilutions vary by application. For IHC applications, typical dilutions range from 1:100 to 1:300 . For ELISA, a much higher dilution of approximately 1:20000 may be optimal . For ICC/IF applications, dilutions between 1:50 and 1:200 are commonly used . These recommendations provide starting points, but optimal dilutions should be determined experimentally for each specific application and sample type.

How do researchers distinguish between true MAP3K10 signal and non-specific binding?

Distinguishing specific from non-specific binding requires rigorous validation. A comprehensive approach includes: (1) comparing results from multiple antibodies targeting different epitopes of MAP3K10, (2) using positive and negative control tissues known to express or lack MAP3K10, (3) conducting peptide competition assays with the immunizing peptide, and (4) validating molecular weight through Western blotting . The observed molecular weight of 72 kDa versus the calculated 103.7 kDa should be investigated when evaluating specificity .

What considerations are important when selecting between different MAP3K10 antibody binding regions?

MAP3K10 antibodies target various regions, including N-terminal (AA 110-138), C-terminal, and internal domains . The choice of epitope region impacts experimental outcomes in several ways. N-terminal antibodies may detect full-length protein but miss truncated forms. C-terminal antibodies might recognize post-translationally modified versions. Selection should be guided by the research question, considering whether detection of specific domains, isoforms, or modified forms is critical to the study .

How should researchers approach conflicting results between different MAP3K10 antibody clones?

When faced with conflicting results, researchers should systematically: (1) verify each antibody's validation data, including the specific epitope recognized, (2) compare immunogen sequences with the target protein sequence in your experimental system, (3) evaluate whether post-translational modifications might affect epitope recognition, (4) consider knockout or knockdown controls to confirm specificity, and (5) perform technical replicates with standardized protocols to identify variables affecting results .

What are the critical validation steps needed before using a MAP3K10 antibody for a novel application?

Before employing a MAP3K10 antibody in a novel application, researchers should: (1) review existing validation data for other applications, (2) conduct preliminary titration experiments to establish optimal concentration, (3) include positive and negative controls, (4) perform specificity tests such as pre-absorption with immunizing peptide, (5) compare results with alternative detection methods, and (6) validate findings with functional assays that correlate with MAP3K10 activity .

How does sample preparation affect MAP3K10 detection in different experimental contexts?

Sample preparation significantly impacts MAP3K10 detection. For IHC applications, PFA-fixation and paraffin embedding are compatible with many MAP3K10 antibodies . For ICC/IF, Triton X-100 permeabilization after PFA fixation has been successfully used . For protein extraction in Western blotting, buffer composition (particularly phosphatase inhibitors) is critical if studying phosphorylated states of MAP3K10. The reducing conditions during sample preparation may affect epitope accessibility and should be optimized .

What are the recommended protocols for detecting MAP3K10 in immunohistochemistry?

For optimal IHC detection of MAP3K10, researchers should: (1) prepare tissue sections at 4-6 μm thickness for paraffin-embedded samples, (2) perform antigen retrieval (typically heat-mediated in citrate buffer pH 6.0), (3) block endogenous peroxidase activity followed by protein blocking, (4) apply primary antibody at 1:100-1:300 dilution , (5) incubate at 4°C overnight, (6) apply appropriate secondary antibody system, and (7) develop using DAB or other chromogen. Human brain tissue has been successfully used for validating MAP3K10 antibodies in IHC applications .

How should researchers optimize Western blotting protocols for MAP3K10 detection?

For effective Western blot detection: (1) prepare samples with phosphatase inhibitors if phosphorylation states are relevant, (2) load adequate protein (typically 20-50 μg per lane), (3) use 8-10% SDS-PAGE gels to properly resolve the protein (observed at 72 kDa, calculated at 103.7 kDa) , (4) transfer to PVDF or nitrocellulose membrane at controlled voltage, (5) block with 5% non-fat milk or BSA, (6) apply primary antibody at approximately 1:1000 dilution , (7) incubate overnight at 4°C, and (8) use appropriate HRP-conjugated secondary antibody followed by ECL detection.

What controls are essential when using MAP3K10 antibodies in research?

Essential controls include: (1) positive tissue/cell controls known to express MAP3K10 (such as brain tissue or A431 cells) , (2) negative controls where primary antibody is omitted, (3) isotype controls using non-specific IgG from the same species as the primary antibody, (4) peptide competition controls where available, and (5) ideally, genetic controls such as tissues/cells with MAP3K10 knockdown or knockout. These controls collectively validate specificity and minimize false positive/negative results .

What is the significance of the discrepancy between observed and calculated molecular weights for MAP3K10?

The observed molecular weight of MAP3K10 (72 kDa) differs significantly from its calculated weight (103.7 kDa) . This discrepancy could result from: (1) post-translational modifications affecting protein mobility, (2) detection of specific isoforms or splice variants, (3) proteolytic processing of the full-length protein, or (4) technical factors in SDS-PAGE. Researchers should address this discrepancy by using multiple antibodies targeting different epitopes and correlating findings with mRNA expression data or other protein detection methods .

How can MAP3K10 antibodies be used in multi-label immunofluorescence experiments?

For multi-label immunofluorescence: (1) verify primary antibodies are from different host species or use directly conjugated antibodies, (2) establish optimal fixation conditions (PFA fixation with Triton X-100 permeabilization has been validated) , (3) implement sequential staining if antibodies have cross-reactivity concerns, (4) use appropriate blocking to minimize non-specific binding, (5) perform careful titration of each antibody, (6) include single-label controls to assess bleed-through, and (7) acquire images with proper filter sets to avoid spectral overlap.

How should researchers address weak or absent MAP3K10 signal in immunostaining applications?

When encountering weak or absent signals: (1) verify tissue/cell expression levels through database mining or other detection methods, (2) optimize antigen retrieval methods (test both heat-mediated and enzymatic approaches), (3) increase antibody concentration incrementally, (4) extend primary antibody incubation time (overnight at 4°C), (5) test alternative detection systems with higher sensitivity, (6) ensure samples were properly preserved to maintain epitope integrity, and (7) consider whether the epitope might be masked by protein interactions or post-translational modifications .

What strategies can resolve high background when using MAP3K10 antibodies?

To reduce high background: (1) optimize blocking conditions (test BSA, normal serum, or commercial blockers), (2) increase blocking time, (3) dilute primary antibody further, (4) reduce primary and secondary antibody incubation times, (5) add 0.1-0.3% Triton X-100 to antibody diluent to reduce non-specific binding, (6) increase washing duration and frequency, (7) pre-absorb secondary antibodies with tissue powder from the species being studied, and (8) consider using more specific detection systems such as polymer-based rather than avidin-biotin methods .

How can researchers validate MAP3K10 antibody specificity in their experimental system?

To validate specificity: (1) perform Western blotting to confirm the detected protein is of the expected size, (2) test multiple antibodies targeting different MAP3K10 epitopes, (3) include known positive and negative control samples, (4) conduct peptide competition assays when possible, (5) verify results with gene silencing approaches (siRNA/shRNA), (6) correlate protein detection with mRNA expression data, and (7) assess functional readouts downstream of MAP3K10 to confirm biological relevance of detection .

What approaches help distinguish MAP3K10 from other closely related kinase family members?

To distinguish MAP3K10 from related kinases: (1) select antibodies raised against unique regions with minimal sequence homology to other family members, (2) perform rigorous specificity testing with recombinant proteins of related kinases, (3) use cells/tissues with differential expression of MAP3K10 versus related kinases, (4) combine antibody detection with functional assays specific to MAP3K10, (5) validate findings with genetic approaches targeting MAP3K10 specifically, and (6) perform careful bioinformatic analysis of the epitope regions to identify potential cross-reactivity .

How should unexpected bands in Western blotting with MAP3K10 antibodies be interpreted?

When encountering unexpected bands: (1) compare observed versus expected molecular weight (72 kDa observed vs. 103.7 kDa calculated) , (2) assess whether bands might represent isoforms, post-translational modifications, or degradation products, (3) verify specificity through peptide competition, (4) test alternative antibodies targeting different epitopes, (5) confirm findings in multiple cell/tissue types, (6) correlate with mRNA expression data for potential splice variants, and (7) consider cross-reactivity with structurally similar proteins .

How can MAP3K10 antibodies contribute to studying neurodegenerative diseases?

MAP3K10 antibodies can advance neurodegeneration research by: (1) analyzing expression patterns in normal versus diseased brain tissues, (2) studying kinase activation in stress response pathways implicated in neurodegeneration, (3) investigating co-localization with disease-associated proteins, (4) examining MAP3K10 involvement in neuronal apoptotic pathways, (5) assessing kinase activity changes during disease progression, and (6) evaluating potential as a therapeutic target. Human brain tissue has been validated for MAP3K10 antibody applications, making such studies feasible .

What considerations are important when using MAP3K10 antibodies for protein-protein interaction studies?

When studying protein interactions: (1) select antibodies that don't interfere with known interaction domains, (2) verify antibodies work in non-denaturing conditions for co-immunoprecipitation, (3) include appropriate controls (IgG control, input controls), (4) validate interactions through reciprocal IP approaches, (5) consider epitope availability in protein complexes, (6) optimize lysis conditions to preserve interactions while ensuring efficient extraction, and (7) confirm findings with alternative methods such as proximity ligation assays .

How can MAP3K10 antibodies be utilized in high-throughput screening or multiplex assays?

For high-throughput applications: (1) rigorously validate antibody specificity to minimize false positives/negatives, (2) determine optimal concentration for maximum signal-to-noise ratio, (3) test compatibility with automated platforms, (4) assess performance in multiplexed formats for potential cross-reactivity, (5) establish reproducible quantification methods, (6) develop appropriate normalization strategies, and (7) include well-characterized controls to ensure consistent performance across batches. These approaches enable MAP3K10 analysis across large sample sets or in combination with other biomarkers .

What role can MAP3K10 antibodies play in investigating cancer signaling pathways?

MAP3K10 antibodies can illuminate cancer signaling by: (1) profiling expression across tumor types and grades, (2) correlating with clinical outcomes and therapeutic responses, (3) investigating activation states in response to oncogenic stimuli, (4) studying interaction with known oncogenes and tumor suppressors, (5) monitoring kinase activity during treatment response and resistance development, and (6) evaluating potential as a biomarker. Human epidermoid carcinoma cell lines (A431) have been validated for MAP3K10 antibody applications, providing a foundation for cancer studies .

How should researchers approach studying phosphorylation states of MAP3K10?

To study MAP3K10 phosphorylation: (1) use phospho-specific antibodies when available, (2) incorporate phosphatase inhibitors during sample preparation, (3) compare results with and without phosphatase treatment, (4) utilize Phos-tag or similar technologies for mobility shift detection, (5) correlate with kinase activity assays, (6) consider temporal dynamics of phosphorylation in experimental design, and (7) validate findings with mass spectrometry or other direct phosphorylation detection methods. This approach provides insights into MAP3K10 regulation and activation in signaling cascades .