MTMR12 Antibody, HRP conjugated

What is MTMR12 and what is its biological significance in experimental models?

MTMR12 (also known as 3-PAP or PIP3AP) functions as an adaptor subunit in a complex with active phosphatidylinositol 3-phosphate (PtdIns(3)P) 3-phosphatase. The protein plays a critical role in regulating membrane-anchored phosphatidylinositides, which are essential for diverse cellular processes . As an experimental target, MTMR12 is particularly valuable for investigating phosphoinositide metabolism and cellular signaling pathways.

MTMR12 shows significant structural similarity to MTM1 and MTMR1, all belonging to the same phylogenetic subgroup. These proteins share 3-phosphatase activity towards PI3P and PI(3,5)P2, making them important regulators of phospholipid balance in cells . In research contexts, MTMR12 is frequently studied alongside MTM1 due to their functional relationship, particularly in muscle tissue where their interaction is critical.

What structural domains characterize MTMR12 and how do they influence antibody development?

MTMR12 contains several functional domains that influence epitope selection for antibody development:

• GRAM domain: An N-terminal lipid or protein interacting domain

• RID: Putative membrane targeting motif

• PTP/DSP: Phosphatase domain

• SID: Protein-protein interacting domain

• CC: Coiled-coil domain

• PDZB: PDZ binding site

Researchers developing or selecting MTMR12 antibodies should consider which domain they wish to target. The search results indicate that available antibodies target different regions, including middle regions (amino acids 188-318) and other segments . The domain-specific targeting affects not only antibody specificity but also which protein interactions may be detected or disrupted in experimental systems.

How does an HRP-conjugated MTMR12 antibody differ from unconjugated versions in experimental applications?

HRP-conjugated MTMR12 antibodies have horseradish peroxidase directly linked to the antibody molecule, providing a convenient one-step detection system compared to unconjugated versions. The primary advantages in experimental workflows include:

• Elimination of secondary antibody requirements, reducing protocol time and potential cross-reactivity

• Enhanced sensitivity for detecting low-abundance MTMR12 protein

• Compatibility with various chemiluminescent, colorimetric, and chemifluorescent substrates

• More consistent signal generation due to defined HRP:antibody ratio

The available MTMR12 HRP-conjugated antibody (CSB-PA880158LB01HU-50) is a rabbit polyclonal antibody generated against recombinant Human MTMR12 protein (amino acids 188-318) . When designing experiments, researchers should consider that while HRP conjugation offers detection advantages, it may affect antibody binding kinetics or accessibility to certain epitopes compared to unconjugated versions.

What validated applications exist for MTMR12 HRP-conjugated antibodies, and what are the optimal protocols?

Based on the search results, MTMR12 antibodies have been validated primarily for Western blotting (WB) . While specific protocols for the HRP-conjugated version are not detailed in the search results, a standard Western blot methodology for MTMR12 detection can be adapted as follows:

• Sample preparation: Extract proteins from tissues or cells using standard lysis buffers containing protease inhibitors

• Protein separation: Resolve 10-30 μg of protein lysate on SDS-PAGE (8-10% gel recommended for detecting the 86 kDa MTMR12 protein)

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane

• Blocking: Block membrane with 50% TBS, 0.1% Tween 20, and 50% Odyssey blocking buffer for 60 minutes at room temperature

• Primary antibody: Dilute HRP-conjugated MTMR12 antibody (typically 1:1000-1:5000) in TBS, 0.1% Tween 20, and 1% milk; incubate for 60 minutes at room temperature or overnight at 4°C

• Washing: Wash membrane 3-5 times with TBST

• Detection: Apply appropriate chemiluminescent substrate and image

Note that when using the HRP-conjugated antibody, the secondary antibody incubation step is eliminated, streamlining the protocol.

How can researchers validate the specificity of MTMR12 antibodies in their experimental systems?

Validating antibody specificity is crucial for generating reliable data. For MTMR12 antibodies, implement these methodological approaches:

• Positive controls: Use cell lysates known to express MTMR12, such as skeletal muscle tissue or C2C12 myoblasts

• Knockdown validation: Perform siRNA-mediated knockdown of MTMR12 in relevant cell lines (e.g., C2C12 myoblasts) and confirm reduced antibody signal by Western blot

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (for the HRP-conjugated antibody, this would be the recombinant Human MTMR12 protein fragment, amino acids 188-318) before application to the membrane

• Cross-species validation: The MTMR12 antibody described in search result shows predicted reactivity across multiple species including human, mouse, rat, cow, dog, horse, guinea pig, rabbit, and zebrafish. Testing the antibody in different species can help confirm specificity for conserved epitopes.

• Molecular weight verification: Confirm that the detected band appears at the expected molecular weight of 86 kDa

What experimental considerations are critical when studying MTMR12-MTM1 protein interactions?

The interaction between MTMR12 and MTM1 is physiologically significant and requires specific experimental approaches to study effectively:

• Co-immunoprecipitation methodology: When designing co-IP experiments to study MTMR12-MTM1 interactions, consider that mutations in the GRAM or RID domains of MTM1 have been shown to disrupt the interactions between MTM1 and MTMR12 . This suggests these domains are critical for the protein-protein interaction.

• Expression level considerations: Research shows that MTMR12 knockdown leads to decreased MTM1 protein levels, suggesting a stabilizing effect of MTMR12 on MTM1 . This interdependence must be accounted for when interpreting results:

  Experimental Condition	Effect on Protein Levels
  MTMR12 siRNA knockdown in C2C12 myoblasts	Decreased MTM1 protein levels
  MTMR12 siRNA knockdown in myotubes	Decreased MTM1 and increased desmin levels
  MTM1 knockout mice	Highly reduced MTMR12 protein levels in skeletal muscle
  MTM1 siRNA knockdown in C2C12 cells	No reduction in MTMR12 protein

• Developmental timing: The relationship between MTMR12 and MTM1 has been observed in both pre-symptomatic (2 weeks) and symptomatic stages (5 weeks) in knockout mouse models, indicating their interaction is developmentally regulated .

How can researchers interpret differences in MTMR12 expression across differentiation stages in muscle cells?

MTMR12 expression analysis in muscle differentiation requires careful experimental design and interpretation:

• Differentiation markers: When analyzing MTMR12 expression during myoblast differentiation, researchers should concurrently examine established markers such as myogenin and desmin. Research has shown that while MTMR12 knockdown affects desmin levels, it doesn't significantly impact myogenin expression , suggesting pathway-specific effects.

• Temporal analysis protocol:

  • Establish C2C12 myoblast cultures

  • Induce differentiation by switching to low-serum medium

  • Collect samples at defined time points (typically days 0, 2, 4, 6)

  • Process parallel samples for RNA (qPCR) and protein (Western blot) analysis

  • Use MTMR12 HRP-conjugated antibody for protein detection

  • Normalize expression to appropriate housekeeping controls (α-actinin has been used successfully)

• Functional correlation: Link MTMR12 expression changes to functional outcomes using cellular assays that measure:

  • Myotube formation (fusion index)

  • Phosphoinositide levels (using specific probes)

  • Cell survival and proliferation metrics

What methodological approaches are effective for studying MTMR12 in disease models related to myotubular myopathy?

Myotubular myopathy models provide valuable insights into MTMR12 function. The following methodological framework can guide research in this area:

• Animal model selection:

  • Mtm1 knockout mice show reduced MTMR12 protein levels in skeletal muscle and can serve as models to study MTMR12 compensation mechanisms

  • Research has demonstrated that recombinant AAV9 vectors expressing MTMR2 (a protein closely related to MTMR12) can rescue muscle pathology in myotubular myopathy models

• Therapeutic intervention assessment:

  • Intramuscular administration protocol: Inject 10 μl of viral vectors (2-3 × 10^10 vg) into the tibialis anterior muscle of 2-week-old animals

  • Systemic administration: Use intravenous injection at a dose of 2.4 × 10^14 vg/kg of body weight

  • Tissue collection: Harvest tissues after 2-4 weeks, quickly freeze in isopentane cooled with liquid nitrogen and store at -80°C

• Protein complex analysis:

  • When studying MTMR12-MTM1 complexes, consider that:

    • Mutations affecting MTM1 stability also result in reduced MTMR12 levels

    • The interaction can be disrupted by specific domain mutations, particularly in GRAM or RID domains

  • These insights can inform experimental designs targeting therapeutic approaches

How does the phosphatase activity of MTMR12 compare to other myotubularin family members in experimental systems?

Understanding the enzymatic properties of MTMR12 relative to other family members requires specific experimental approaches:

• Comparative phosphatase activity measurement:

  • MTMR12 belongs to the same phylogenetic subgroup as myotubularin (MTM1) and MTMR1

  • These proteins share 3-phosphatase activity towards PI3P and PI(3,5)P2

  • When designing experiments to measure phosphatase activity, researchers should:

    • Use purified recombinant proteins

    • Employ specific phosphoinositide substrates

    • Monitor product formation using HPLC, mass spectrometry, or colorimetric assays

• Substrate specificity determination:

  • Research indicates that MTMR12 functions as an adaptor subunit in a complex with an active PtdIns(3)P 3-phosphatase

  • Experimental designs should incorporate multiple substrates to determine specificity profiles:

    • PI3P

    • PI(3,5)P2

    • Other phosphoinositides as negative controls

• Complex formation effects on activity:

  • MTMR12 can form complexes with active phosphatases like MTM1

  • To study these interactions experimentally:

    • Express tagged versions of both proteins

    • Perform co-immunoprecipitation followed by phosphatase activity assays

    • Compare activity of individual proteins versus complexes

What are common technical challenges when using MTMR12 HRP-conjugated antibodies and how can they be resolved?

Researchers may encounter several challenges when working with MTMR12 HRP-conjugated antibodies:

• High background signal:

  • Problem: Non-specific HRP activity causing elevated background

  • Solutions:

    • Increase blocking time and concentration (use 50% Odyssey blocking buffer with 50% TBS and 0.1% Tween 20)

    • Reduce antibody concentration

    • Add 1-5% milk or BSA to antibody diluent

    • Increase washing steps (5-6 washes of 5-10 minutes each)

• Multiple bands or unexpected molecular weights:

  • Problem: Detection of splice variants, degradation products, or non-specific binding

  • Solutions:

    • Verify the expected 86 kDa molecular weight for MTMR12

    • Check for known splice variants (MTMR12 has alternative splice variants)

    • Use fresh samples with protease inhibitors to minimize degradation

    • Perform peptide competition assays to identify specific bands

• Weak or absent signal:

  • Problem: Low antibody affinity, low target expression, or inefficient transfer

  • Solutions:

    • Increase protein loading (up to 30-50 μg)

    • Optimize antibody concentration

    • Use enhanced chemiluminescent substrates

    • Extend exposure time

    • Consider alternative epitopes (the HRP-conjugated antibody targets amino acids 188-318)

How can researchers accurately quantify changes in MTMR12 expression following experimental manipulations?

Accurate quantification of MTMR12 expression changes requires careful methodological consideration:

• Western blot quantification protocol:

  • Use appropriate loading controls (α-actinin, β-actin, or GAPDH have been successfully used)

  • Ensure signal is within linear detection range

  • Perform at least three independent experiments

  • Normalize MTMR12 signal to loading control

  • Express results as fold change relative to control condition

• Statistical analysis approach:

  • Apply appropriate statistical tests (t-test for two conditions, ANOVA for multiple conditions)

  • Report p-values (significant changes have been reported at P<0.05)

  • Include error bars representing standard deviation or standard error

  • Present normalized quantification in histogram format

• Validation across methods:

  • Complement protein expression data with mRNA analysis

  • Consider immunofluorescence to assess cellular localization changes

  • For knockdown experiments, verify reduction at both mRNA and protein levels

What control experiments are essential when investigating MTMR12-MTM1 interactions in cellular models?

When studying the relationship between MTMR12 and MTM1, several critical controls must be incorporated:

• Expression level controls:

  • When manipulating one protein, monitor effects on the other:

    • MTMR12 knockdown reduces MTM1 protein levels

    • MTM1 knockout reduces MTMR12 levels in muscle tissue

    • MTM1 knockdown in cell lines may not affect MTMR12 levels

  • These differential effects must be considered when interpreting results

• Domain mutation controls:

  • Include MTM1 constructs with mutations in GRAM or RID domains as negative controls for interaction

  • Wild-type MTM1 should be used as a positive control

  • When testing interactions with co-immunoprecipitation, include an unrelated protein as a negative control

• Cell type considerations:

  • The MTMR12-MTM1 relationship may vary by cell type

  • Include multiple relevant cell types (myoblasts, myotubes, non-muscle cells)

  • In knockdown experiments, include rescue conditions by re-expressing the target protein

These methodological controls ensure that observed changes in MTMR12-MTM1 interactions are specific and physiologically relevant, rather than artifacts of experimental manipulation or cell-specific effects.

How can MTMR12 antibodies be utilized in multiplexed imaging approaches for tissue analysis?

While traditional applications of MTMR12 antibodies focus on Western blotting, advanced imaging techniques offer new research possibilities:

• Multiplex immunofluorescence protocol development:

  • The HRP-conjugated MTMR12 antibody can be used with tyramide signal amplification (TSA)

  • This approach allows for sequential staining of multiple proteins on the same tissue section

  • Workflow:

    1. Apply MTMR12-HRP antibody (diluted 1:100-1:500)

    2. Develop with tyramide-conjugated fluorophore

    3. Inactivate HRP with hydrogen peroxide

    4. Apply next primary antibody

    5. Repeat steps 2-4 for additional targets

• Co-localization analysis methodology:

  • For co-localization studies with MTM1, researchers should:

    • Use paraffin-embedded or frozen muscle sections from relevant models

    • Apply MTMR12-HRP antibody with TSA-fluorophore development

    • Counter-stain with antibodies against MTM1 and other interacting proteins

    • Image using confocal microscopy with appropriate controls for spectral overlap

    • Analyze using co-localization algorithms (e.g., Pearson's correlation coefficient)

• Tissue-specific expression profiling:

  • MTMR12 antibodies can be used to create tissue-specific expression maps

  • This approach requires careful validation of antibody specificity across tissues

  • Include appropriate positive and negative control tissues

What methodological considerations are important when studying MTMR12 in gene therapy approaches for myopathies?

Gene therapy research for myopathies requires specific considerations when investigating MTMR12:

• Vector design methodology:

  • Research has demonstrated that AAV9 vectors expressing MTMR2 (related to MTMR12) can rescue muscle pathology in myotubular myopathy models

  • When designing MTMR12-based vectors:

    • Consider using muscle-specific promoters (human desmin promoter has been used successfully)

    • Clone the complete MTMR12 coding sequence into AAV2 plasmid backbone

    • Package AAV2-ITR recombinant genomes into AAV9 capsids for optimal muscle targeting

• Delivery protocol optimization:

  • For localized effects: Intramuscular injection of 10 μl (2-3 × 10^10 vg)

  • For systemic treatment: Intravenous injection of 2.4 × 10^14 vg/kg

  • Optimal timing: Treatment at 2 weeks of age in mouse models

  • Evaluation timepoint: Tissue analysis 2-4 weeks post-injection

• Therapeutic efficacy assessment:

  • Measure MTMR12 and MTM1 protein levels by Western blot

  • Assess histopathological improvements in muscle tissue

  • Evaluate functional outcomes through strength testing and survival analysis

  • Compare efficacy to established treatments (e.g., MTM1 gene therapy)