MTMR9 Antibody, Biotin conjugated

What is MTMR9 and why is it a significant research target?

MTMR9 (Myotubularin-related protein 9) is a 549 amino acid protein belonging to the protein-tyrosine phosphatase family and non-receptor class myotubularin subfamily. Unlike other members of the myotubularin family, MTMR9 lacks a dual-specificity phosphatase domain and functions as a pseudophosphatase. It contains a double-helical motif similar to the SET interaction domain and is believed to play a role in cell proliferation control . MTMR9 forms functional heteromeric complexes with catalytically active myotubularins (particularly MTMR6, MTMR7, and MTMR8), enhancing their enzymatic activity, stability, and cellular functions . This protein is widely expressed in various tissues, including brain, making it a significant target for studying phosphoinositide signaling and regulation.

What are the advantages of using biotin-conjugated MTMR9 antibodies in research?

Biotin-conjugated MTMR9 antibodies offer several methodological advantages:

• Enhanced sensitivity through biotin-streptavidin interaction, which provides signal amplification

• Versatility in detection systems as biotin can be detected with various streptavidin-conjugated reporters

• Compatibility with multi-color immunofluorescence studies as the biotin-streptavidin system can be combined with directly labeled antibodies

• Reduced background in tissue sections due to the high affinity and specificity of the biotin-streptavidin interaction

• Stability of conjugate, as biotin conjugation typically preserves antibody functionality better than some direct fluorophore conjugations

How should I select the appropriate MTMR9 antibody based on epitope recognition?

Selection should be guided by the specific region of MTMR9 you wish to target and your experimental context:

When studying MTMR9-MTMR6 interactions, antibodies targeting AA 322-549 may be particularly useful as this region contains domains involved in protein-protein interactions that affect enzymatic activity .

What controls should be included when using biotin-conjugated MTMR9 antibodies?

A comprehensive control strategy should include:

• Positive control: Cell lines or tissues with known MTMR9 expression (human brain tissue extracts are recommended)

• Negative control: Samples from MTMR9 knockout models or cells treated with MTMR9-specific siRNA

• Isotype control: Biotin-conjugated isotype-matched immunoglobulin (same host species and IgG subclass) to detect non-specific binding

• Blocking control: Pre-incubation with the immunizing peptide to confirm specificity

• Endogenous biotin blocking: Treatment with avidin/biotin blocking kit when working with tissues containing endogenous biotin

• Secondary-only control: When using secondary detection methods to assess background

These controls help distinguish true signals from artifacts, particularly important when investigating the subtle co-localization patterns of MTMR9 with binding partners like MTMR6 .

How should samples be prepared for optimal detection of MTMR9 using biotin-conjugated antibodies?

For optimal detection in different applications:

For Western Blotting:

• Lyse cells in buffer containing phosphatase inhibitors since MTMR9 is involved in phosphoinositide metabolism

• Use fresh samples when possible; avoid repeated freeze-thaw cycles

• Recommended dilution range: 1:1000-1:2000

For Immunofluorescence/Immunohistochemistry:

• Fixation: 4% paraformaldehyde is preferred for preserving protein epitopes

• Permeabilization: 0.1-0.5% Triton X-100 for cytoplasmic proteins like MTMR9

• Blocking: Use BSA or serum plus biotin blocking reagents to minimize background

• Recommended dilution range: 1:50-1:200

• Note: When examining MTMR9-MTMR6 co-localization, maintaining physiological protein levels is crucial to avoid artifacts from overexpression

For ELISA:

• Follow sandwich ELISA format for quantitation

• Use biotin-conjugated detection antibody followed by streptavidin-HRP

• Sample types validated: serum, plasma, cell culture supernatants

What is the optimal protocol for detecting MTMR9-MTMR6 interactions using biotin-conjugated antibodies?

For investigating MTMR9-MTMR6 interactions:

• Co-immunoprecipitation approach:

  • Lyse cells in non-denaturing buffer containing protease inhibitors

  • Perform initial IP with anti-MTMR6 antibodies

  • Detect co-precipitated MTMR9 using biotin-conjugated anti-MTMR9 antibody

  • Visualize with streptavidin-HRP or fluorescently labeled streptavidin

  • Recommended dilution: 1:100-1:500 for detection

• Proximity Ligation Assay (PLA):

  • Fix cells in 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Incubate with primary antibodies targeting MTMR9 and MTMR6

  • Use biotin-conjugated anti-MTMR9 with streptavidin-linked PLA probe

  • Perform ligation and amplification according to PLA protocol

  • This technique allows visualization of endogenous protein interactions in situ

Interactions should be validated under conditions that preserve native protein conformations, as heteromer formation substantially affects both proteins' stability .

How can I validate the specificity of biotin-conjugated MTMR9 antibodies?

Validating antibody specificity requires a multi-faceted approach:

• Immunoblotting with recombinant proteins:

  • Test against both recombinant MTMR9 and related family members (MTMR6-8)

  • Verify single band at expected molecular weight (~63 kDa for MTMR9)

• siRNA/shRNA knockdown validation:

  • Compare signal intensity between control and MTMR9-depleted samples

  • Expect 20-40% reduction with MTMR9 siRNA alone, and up to 90% reduction when both MTMR9 and binding partners are targeted

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Expected result: significant reduction or elimination of specific signal

• Cross-reactivity testing:

  • Test against closely related proteins, particularly other myotubularin family members

  • Verify antibody does not detect MTMR8 despite similar nomenclature in some databases

• Comparison with multiple antibodies:

  • Use antibodies targeting different epitopes to confirm consistent localization patterns

What are the common challenges with biotin-conjugated antibodies in tissue samples and how can they be overcome?

Several challenges may arise when using biotin-conjugated antibodies in tissue samples:

For perinuclear staining patterns typical of MTMR9, carefully optimize fixation protocols to preserve subcellular localization patterns while maintaining antigenicity .

How can biotin-conjugated MTMR9 antibodies be utilized to study the regulatory role of MTMR9 in phosphoinositide metabolism?

To investigate MTMR9's regulatory effects on phosphoinositide metabolism:

• Biochemical approaches:

  • Perform lipid binding assays to assess how MTMR9 affects MTMR6 binding to phospholipids

  • Use biotin-conjugated MTMR9 antibodies in pull-down experiments followed by lipid extraction and analysis

  • Quantify specific phosphoinositides (PtdIns3P, PtdIns3,5P₂) in samples with normal or altered MTMR9 expression

• Cellular localization studies:

  • Use biotin-conjugated MTMR9 antibodies with fluorescent phosphoinositide sensors

  • Co-staining with markers for specific subcellular compartments where phosphoinositide metabolism occurs

  • Implement super-resolution microscopy techniques to visualize precise co-localization patterns

• Enzymatic activity assessment:

  • Compare 3-phosphatase activity of MTMR6 alone versus MTMR6-MTMR9 complex (up to 6-fold increase expected)

  • In the presence of phosphatidylserine liposomes, expect up to 84-fold increase in activity when MTMR9 is present

• Protein stability analysis:

  • Monitor MTMR6 half-life (increases from ~40 min to 4 hours when complexed with MTMR9)

  • Quantitative immunofluorescence to measure protein levels after cycloheximide treatment

How can biotin-conjugated MTMR9 antibodies be employed in studying the role of MTMR9 in apoptotic pathways?

For investigating MTMR9's role in apoptosis regulation:

• Apoptosis induction studies:

  • Treat cells with etoposide or other apoptosis inducers

  • Compare cells with normal, overexpressed, or depleted MTMR9 levels

  • Use biotin-conjugated MTMR9 antibodies to quantify protein levels before and during apoptosis

  • Expect decreased etoposide-induced apoptosis when MTMR9 is co-expressed with MTMR6

• Time-course experiments:

  • Monitor MTMR9 and MTMR6 levels at different time points after apoptosis induction

  • Utilize biotin-conjugated antibodies in flow cytometry to correlate MTMR9 levels with apoptotic markers

• Subcellular redistribution analysis:

  • Track potential changes in MTMR9 localization during apoptosis

  • Use confocal microscopy with biotin-conjugated MTMR9 antibodies and streptavidin-fluorophore detection

  • Co-stain with markers for apoptotic structures

• Protein-protein interaction dynamics:

  • Analyze how MTMR9-MTMR6 interaction changes during apoptosis

  • Implement FRET-based approaches using biotin-conjugated antibodies and appropriate fluorescent streptavidin conjugates

Research has shown that RNAi of both MTMR9 and MTMR6 leads to increased cell death in response to etoposide compared to MTMR6 knockdown alone .

How should researchers interpret differences in MTMR9 detection across various experimental platforms?

When interpreting MTMR9 detection results across different platforms:

• Western blot vs. immunofluorescence discrepancies:

  • WB detects denatured proteins, while IF detects native conformations

  • MTMR9 antibodies may have conformation-dependent epitope recognition

  • Some epitopes may be masked by heteromer formation with MTMR6/7/8

  • Expected perinuclear localization in IF should correlate with proper detection in WB

• ELISA vs. other techniques:

  • Sandwich ELISA may detect different epitopes simultaneously

  • Quantitative differences may reflect heteromer dissociation during sample processing

  • When using biotin-conjugated antibodies in sandwich ELISA, ensure primary capture antibody targets a different epitope

• Species-specific considerations:

  • Human MTMR9: All testing methods supported

  • Mouse/Rat MTMR9: May require specific antibody validation as cross-reactivity varies

  • Amino acid sequence conservation should be verified for cross-species applications

• Technical validation metrics:

  • Signal-to-noise ratio >3:1 indicates reliable detection

  • Coefficient of variation <15% for quantitative applications

  • Linear dynamic range should be established for each application

What are the most effective strategies for multiplex detection incorporating biotin-conjugated MTMR9 antibodies?

For effective multiplex detection strategies:

• Sequential detection approach:

  • Complete biotin-streptavidin detection workflow first

  • Block remaining biotin binding sites

  • Proceed with directly labeled antibodies for other targets

  • Particularly useful for co-localization studies of MTMR9 with MTMR6

• Tyramide signal amplification:

  • Use biotin-conjugated MTMR9 antibody with HRP-streptavidin

  • Develop with tyramide-fluorophore

  • Heat-inactivate HRP

  • Proceed with next antibody

  • Enables detection of low-abundance interactions

• Spectral unmixing techniques:

  • Use streptavidin conjugates with narrow emission spectra

  • Apply spectral imaging and computational unmixing

  • Allows discrimination of closely overlapping fluorophores

• Quantum dot-based multiplexing:

  • Utilize streptavidin-conjugated quantum dots with narrow emission profiles

  • Different sized quantum dots can be excited with single wavelength

  • Particularly valuable for studying MTMR9 interactions with multiple partners simultaneously

When studying MTMR9-MTMR6 complexes, consider the co-dependent stability of these proteins when designing experimental workflows .

How might biotin-conjugated MTMR9 antibodies contribute to understanding disease mechanisms?

Biotin-conjugated MTMR9 antibodies can advance understanding of disease mechanisms through:

• Cancer research applications:

  • MTMR9's role in cell proliferation control suggests oncogenic potential

  • Biotin-conjugated antibodies enable sensitive detection of expression changes in tumor samples

  • Correlation of MTMR9 levels with B-cell chronic lymphoid leukemia resistance to apoptosis

  • Potential therapeutic target assessment through antibody-based screening platforms

• Neurodegenerative disease investigations:

  • MTMR9 is expressed in brain tissue

  • Phosphoinositide dysregulation occurs in various neurodegenerative conditions

  • Biotin-conjugated antibodies facilitate multiplexed analysis with neuronal markers

• Metabolic disorder studies:

  • MTMR9's interaction with lipid metabolism pathways

  • Potential links to insulin signaling and metabolic syndrome

  • Antibody-based screens for identifying regulatory pathways

• Tissue-specific expression profiling:

  • Biotin-conjugated antibodies enable high-sensitivity tissue microarray analysis

  • Correlation of expression patterns with disease progression markers

  • Identification of tissue-specific heteromeric complexes with therapeutic potential

What novel methodological approaches could enhance MTMR9 protein interaction studies using biotin-conjugated antibodies?

Innovative approaches for MTMR9 interaction studies include:

• Proximity-dependent labeling techniques:

  • BioID or TurboID fusion with MTMR9

  • Detection of biotinylated proteins using anti-biotin antibodies

  • Identification of transient MTMR9 interactors beyond known MTMR6/7/8 partners

  • Maps the complete MTMR9 protein interaction network

• Single-molecule imaging:

  • Use biotin-conjugated antibodies with quantum dot-streptavidin for single-molecule tracking

  • Analyze dynamics of MTMR9-MTMR6 interactions in living cells

  • Quantify heteromer formation/dissociation kinetics

• Correlative light-electron microscopy:

  • Primary detection with biotin-conjugated MTMR9 antibodies

  • Secondary detection with gold-conjugated streptavidin

  • High-resolution localization at ultrastructural level

  • Particularly valuable for studying perinuclear localization patterns

• Microfluidic antibody-based assays:

  • Develop chip-based systems for rapid quantification of MTMR9 and binding partners

  • Single-cell analysis of expression heterogeneity

  • Real-time monitoring of interaction dynamics during cellular responses