MTMR9 Antibody, FITC conjugated

What is MTMR9 and why is it significant for research?

MTMR9 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, making it a pseudophosphatase. MTMR9 is expressed in various tissues, including brain, and localizes to the cytoplasm . Its significance lies in its interactions with catalytically active myotubularins (MTMR6, MTMR7, and MTMR8), where it functions as a regulator of their enzymatic activity and substrate specificity . MTMR9 contains a double-helical motif similar to the SET interaction domain and may play a role in cell proliferation control .

What does FITC conjugation mean for an MTMR9 antibody?

FITC conjugation involves crosslinking the MTMR9 antibody with the fluorescein isothiocyanate fluorophore using established protocols . This process creates a directly labeled antibody that fluoresces green when excited with appropriate wavelengths, typically from a blue laser (488 nm). The conjugation eliminates the need for secondary antibodies in immunofluorescence applications, reducing background and simplifying experimental workflows. FITC-conjugated antibodies can be used for various techniques including flow cytometry, immunofluorescence microscopy, and ELISA .

How should FITC-conjugated MTMR9 antibodies be stored to maintain optimal activity?

FITC-conjugated antibodies should be stored between 2°C and 8°C (refrigerated) and protected from prolonged exposure to light, as continuous light exposure causes gradual loss of fluorescence . Most commercial FITC-conjugated antibodies are supplied in phosphate-buffered saline (PBS) containing 0.01% sodium azide as a preservative. It's important to never freeze these antibodies, as freezing can damage the protein structure and the fluorophore conjugation, resulting in reduced activity and increased background . Always centrifuge the product briefly prior to opening the vial to avoid concentration in the cap.

What controls should be included when using MTMR9-FITC antibodies in immunofluorescence studies?

When designing immunofluorescence experiments with MTMR9-FITC antibodies, the following controls should be included:

• Isotype control: A FITC-conjugated antibody of the same isotype (e.g., IgG1 kappa for MTMR9 H-1 antibody) but with irrelevant specificity to assess non-specific binding

• Negative cellular control: Cells known not to express MTMR9 or with MTMR9 knockdown

• Secondary antibody-only control: When using unconjugated primary antibodies in parallel experiments

• Autofluorescence control: Untreated cells to establish baseline cellular autofluorescence

• Positive control: Cells known to express MTMR9 at detectable levels

These controls help distinguish true positive signals from technical artifacts and non-specific binding.

What is the recommended protocol for immunofluorescence staining using MTMR9-FITC antibodies?

A standard protocol for immunofluorescence using MTMR9-FITC antibodies includes:

• Cell preparation: Grow cells on coverslips, fix with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: Treat with 0.5% Triton X-100 in PBS for 10 minutes

• Blocking: Incubate with 5% BSA in PBS, 0.1% Triton X-100 for 30-60 minutes

• Primary antibody: Apply MTMR9-FITC antibody diluted 1:500 in blocking buffer, incubate for 1 hour at 37°C

• Washing: Wash 3 times with PBS, 5 minutes each

• Counterstaining: Apply DAPI (1 μg/ml) for nuclear staining, 5 minutes

• Mounting: Mount with anti-fade mounting medium and seal edges

• Imaging: Visualize using appropriate filter sets for FITC (excitation: 488 nm, emission: 520 nm)

This protocol may require optimization depending on cell type, fixation method, and antibody concentration.

How can one accurately quantify colocalization between MTMR9 and its binding partners using FITC-conjugated antibodies?

Accurate quantification of colocalization between MTMR9-FITC and its binding partners requires:

• Multi-channel imaging:

  • Use MTMR9-FITC antibody for green channel

  • Use spectrally distinct fluorophores (e.g., Alexa 594/647) for binding partners (MTMR6, MTMR7, or MTMR8)

• Image acquisition settings:

  • Capture with sequential scanning to prevent bleed-through

  • Use identical settings for all samples

  • Include single-label controls to establish thresholds

• Quantification methods:

  • Calculate Pearson's correlation coefficient (values from -1 to +1)

  • Determine Manders' overlap coefficient (M1 and M2)

  • Use intensity correlation analysis (ICA)

• Software tools:

  • ImageJ with JACoP plugin

  • CellProfiler

  • Specialized colocalization software

Research has shown that MTMR9 and MTMR6 exhibit significant colocalization in the perinuclear region, with both proteins accumulating in this area . When quantifying such interactions, it's crucial to analyze multiple cells (n>30) across independent experiments to ensure statistical validity.

What approaches can be used to verify MTMR9-FITC antibody specificity in experimental settings?

To verify MTMR9-FITC antibody specificity:

• Western blot validation:

  • Run parallel western blots with the same antibody (non-conjugated version)

  • Verify single band at expected molecular weight (549 amino acids, ~63 kDa)

• Genetic approaches:

  • Perform siRNA knockdown of MTMR9 and confirm reduction in fluorescence signal

  • Use CRISPR/Cas9 knockout cells as negative controls

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide (e.g., MTMR9 AA 322-549)

  • Signal should be significantly reduced compared to non-competed antibody

• Cross-reactivity testing:

  • Test on tissues/cells from different species based on known reactivity

  • Verify expected subcellular localization patterns (primarily cytoplasmic with perinuclear enrichment)

• Multiple antibody validation:

  • Compare staining patterns of different antibodies targeting distinct MTMR9 epitopes

How can MTMR9-FITC antibodies be used to investigate the regulatory role of MTMR9 on phosphoinositide metabolism?

MTMR9-FITC antibodies can be instrumental in investigating phosphoinositide metabolism through:

• Co-immunoprecipitation with imaging validation:

  • Use MTMR9-FITC to visualize MTMR9 localization before and after co-IP

  • Correlate with binding partners (MTMR6/MTMR8) and phosphoinositide distribution

• Substrate specificity analysis:

  • Combine immunofluorescence with phosphoinositide sensors (e.g., PtdIns(3)P, PtdIns(3,5)P₂)

  • Compare wild-type vs. MTMR9-overexpressing or knockdown cells

• Enzyme activity correlation studies:

  • MTMR9 increases MTMR6 activity toward PtdIns(3,5)P₂ by >30-fold but only 2-fold toward PtdIns(3)P

  • MTMR9 increases MTMR8 activity 4-fold toward PtdIns(3)P and 1.4-fold toward PtdIns(3,5)P₂

• Real-time dynamics:

  • Live-cell imaging of MTMR9-GFP with phosphoinositide sensors

  • Correlate with MTMR9-FITC antibody staining in fixed cells to validate constructs

This approach can clarify how MTMR9 determines substrate preference for its binding partners and consequently affects cellular functions.

What experimental approaches can resolve contradictory findings about MTMR9's role in autophagy regulation?

To resolve contradictions in MTMR9's role in autophagy regulation:

• Combinatorial knockdown experiments:

  • Single knockdowns of MTMR8 or MTMR9 showed no effect on p62 levels

  • Double knockdown significantly reduced p62 levels in bafilomycin A1-treated cells

• Systematic time-course analysis:

  • Monitor autophagy markers (LC3-II, p62) at multiple timepoints

  • Combine with MTMR9-FITC staining to correlate protein levels with autophagy dynamics

• Substrate-specific manipulations:

  • Artificially modulate PtdIns(3)P and PtdIns(3,5)P₂ levels independently

  • Determine which substrate is critical for MTMR9's effect on autophagy

• Binding partner analysis:

  • Create binding-deficient MTMR9 mutants that selectively fail to interact with MTMR6 or MTMR8

  • Determine which interaction is critical for autophagy regulation

• Cell-type specific analysis:

  • Compare effects in multiple cell types with varying baseline autophagy levels

  • Use MTMR9-FITC to quantify expression levels in different cell types

The research indicates that MTMR8/MTMR9 complex functions to reduce autophagy, while MTMR6/MTMR9 inhibits apoptosis . These distinct functions appear to be mediated through their differential effects on phosphoinositide substrates.

What are the considerations when designing multiplex experiments using MTMR9-FITC antibodies alongside other fluorophore-conjugated antibodies?

When designing multiplex experiments with MTMR9-FITC antibodies:

• Spectral compatibility:

  • FITC excitation maximum: ~495 nm, emission maximum: ~520 nm

  • Avoid overlapping fluorophores like GFP, Alexa Fluor 488

  • Optimal partners: PE (~565-580 nm), Texas Red (~615 nm), or Alexa Fluor 647 (~668 nm)

• Antibody compatibility:

  • When co-staining for multiple myotubularin family members, use antibodies from different host species

  • For example: Rabbit anti-MTMR6, Mouse anti-MTMR9-FITC, Goat anti-MTMR8

• Signal intensity balancing:

  • FITC signal is typically weaker than newer fluorophores like Alexa dyes

  • May require higher concentration of MTMR9-FITC antibodies relative to other fluorophores

  • Consider using signal amplification methods for FITC channel if necessary

• Sequential staining consideration:

  • When combining with antibodies requiring indirect detection, apply MTMR9-FITC in the last staining step

  • Reduces risk of FITC photobleaching during multiple incubation steps

• Cross-reactivity validation:

  • Always include single-stained controls to verify lack of cross-reactivity

  • Especially important when studying multiple MTMR family members which share sequence homology

How can researchers analyze the oligomerization state of MTMR9 using FITC-conjugated antibodies?

Recent research has shown that MTMR9 forms complexes through its coiled-coil (CC) domain. To analyze these oligomerization states:

• FRET-based approaches:

  • Use MTMR9-FITC antibody as donor

  • Use another MTMR9 antibody conjugated to a FRET-compatible acceptor fluorophore

  • FRET efficiency indicates proximity and can distinguish between dimers and higher-order oligomers

• Fluorescence fluctuation spectroscopy:

  • Apply methods like Number and Brightness (N&B) analysis or Fluorescence Correlation Spectroscopy (FCS)

  • Can determine stoichiometry of MTMR9 complexes in living cells

• Coiled-coil domain analysis:

  • MTMR9-CC forms trimers as shown in recent biophysical studies

  • Compare with MTMR7-CC which forms dimers preferentially

  Protein	Oligomerization Preference	CC Domain Length (residues)	Distance to Core Domain
  MTMR9	Trimers	47	1
  MTMR7	Dimers	30	21

• Chemical crosslinking with FITC detection:

  • Apply membrane-permeable crosslinkers to living cells

  • Perform SDS-PAGE followed by fluorescence scanning

  • MTMR9-FITC antibody can detect different oligomeric states

• Non-denaturing electrophoresis:

  • Perform Blue Native PAGE on cell lysates

  • Use MTMR9-FITC antibody for direct fluorescence detection of native complexes

This combined approach can provide valuable insights into the structural organization of MTMR9 complexes and their dynamic regulation in cells.

What strategies can address weak or absent signal when using MTMR9-FITC antibodies?

When encountering weak or absent signal with MTMR9-FITC antibodies:

• Antibody concentration optimization:

  • Start with manufacturer's recommendation (typically 2 μg/mL)

  • Test concentration range (1-10 μg/mL) to determine optimal signal-to-noise ratio

  • Different applications may require different concentrations

• Antigen retrieval considerations:

  • For fixed samples, test different antigen retrieval methods:

    • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0)

    • Enzymatic retrieval with proteinase K

    • Stronger detergents for permeabilization

• Fixation method evaluation:

  • Compare paraformaldehyde (preserves structure) vs. methanol (better permeabilization)

  • The MTMR9 epitope AA 322-549 may have different accessibility depending on fixation

• Signal amplification options:

  • Anti-FITC antibody followed by fluorophore-conjugated secondary

  • Tyramide signal amplification (TSA)

  • Consider alternative conjugates if FITC signal remains insufficient

• Expression level verification:

  • Confirm MTMR9 expression by RT-PCR or western blot

  • MTMR9 expression might be regulated by its binding partners - co-expression with MTMR6 or MTMR8 enhances stability

How can researchers resolve high background issues when using MTMR9-FITC antibodies?

To resolve high background when using MTMR9-FITC antibodies:

• Blocking optimization:

  • Test different blocking agents:

    • 5% BSA in PBS, 0.1% Triton X-100

    • 10% normal serum from species unrelated to antibody host

    • Commercial blocking solutions

  • Increase blocking time to 1-2 hours

• Antibody dilution series:

  • Test serial dilutions to find optimal concentration

  • Higher concentrations may increase specific signal but also non-specific binding

• Washing protocol enhancement:

  • Increase number of washes (5-6 times)

  • Use PBS with 0.05-0.1% Tween-20

  • Longer wash duration (10-15 minutes per wash)

• Autofluorescence reduction:

  • Treat samples with 0.1% Sudan Black B in 70% ethanol

  • 10 mM CuSO₄ in 50 mM ammonium acetate buffer (pH 5.0)

  • Commercial autofluorescence quenchers

• Tissue/cell-specific considerations:

  • Brain tissue requires special attention due to lipofuscin autofluorescence

  • MTMR9 is expressed in brain tissue, making this consideration relevant

What are the critical considerations when analyzing MTMR9 complex formation in subcellular compartments?

When analyzing MTMR9 complex formation in different compartments:

• Co-localization analysis framework:

  • MTMR9 primarily accumulates in the perinuclear region

  • Use z-stack confocal imaging (0.2-0.3 μm steps) for complete spatial representation

  • Analyze object-based colocalization rather than just pixel overlap

• Dynamic trafficking considerations:

  • Phosphoinositide levels change rapidly in response to stimuli

  • Consider time-lapse imaging or synchronized cell populations

  • MTMR9 complexes may relocalize upon cell stimulation

• Interaction partner-specific patterns:

  • MTMR6/MTMR9 complex: Perinuclear region, ER, ER-Golgi

  • MTMR8/MTMR9 complex: May associate with autophagosome formation sites

  • Differential patterns may explain functional specificity

• Structural domains affecting localization:

  • MTMR9 contains a 47-residue coiled-coil domain positioned only 1 residue from its core domain

  • This structural feature may influence membrane association and subcellular targeting

• Correlation with functional readouts:

  • PtdIns(3)P levels (early endosomes, autophagosome formation sites)

  • PtdIns(3,5)P₂ levels (late endosomes, lysosomes)

  • Autophagy markers (LC3-II puncta, p62 levels)

  • Apoptotic markers (cleaved caspase-3, TUNEL staining)

How do recent findings on MTMR9's intrinsically disordered regions impact antibody selection and experimental design?

Recent research has revealed important insights about MTMR9's structure:

• Intrinsically disordered regions (IDRs):

  • MTMR9 contains uncharacterized IDRs and short linear motifs (SLiMs)

  • These regions may adopt different conformations in different cellular contexts

  • Antibody epitopes within IDRs might be differentially accessible depending on binding partners

• Antibody epitope considerations:

  • Antibodies targeting AA 322-549 region (includes the CC domain) may detect different functional states

  • Comparison of antibodies targeting different epitopes:

    • AA 1-167: suitable for ELISA, IF applications

    • AA 254-428: suitable for WB, IF, IHC(p), ICC applications

    • AA 322-549: suitable for multiple applications including ELISA and IF

• Phase separation potential:

  • IDRs in MTMR9 may participate in phase separation processes

  • This could affect local concentration and detection sensitivity

  • Different fixation methods may preserve or disrupt these condensates

• Experimental design implications:

  • Consider native vs. denaturing conditions carefully

  • Multiple antibodies targeting different regions may provide complementary information

  • Correlation with functional assays becomes even more critical

What methodological approaches can determine how MTMR9 phosphorylation affects its interactions with catalytic partners?

To investigate MTMR9 phosphorylation effects:

• Phosphorylation-specific antibody approach:

  • Generate phospho-specific antibodies against predicted MTMR9 phosphorylation sites

  • Compare localization and complex formation using dual labeling:

    • MTMR9-FITC (total MTMR9)

    • Phospho-MTMR9 specific antibody (different fluorophore)

• Phosphomimetic mutant analysis:

  • Generate S/T→D/E mutations to mimic phosphorylation

  • S/T→A mutations to prevent phosphorylation

  • Compare binding efficiency to MTMR6/MTMR8 using co-IP followed by FITC-antibody detection

• Kinase/phosphatase inhibitor studies:

  • Treat cells with specific kinase inhibitors

  • Monitor changes in MTMR9 complex formation using FITC-antibody

  • Correlate with changes in substrate specificity

• Mass spectrometry validation:

  • Immunoprecipitate MTMR9 and identify phosphorylation sites

  • Correlate site occupancy with binding partner preference

  • Use MTMR9-FITC antibody to verify immunoprecipitation efficiency

• Functional readouts:

  • Monitor phosphoinositide levels (PtdIns(3)P vs. PtdIns(3,5)P₂)

  • Assess autophagy and apoptosis markers

  • Correlate with MTMR9 phosphorylation state

How can MTMR9-FITC antibodies be utilized in investigating MTMR9's role in pathological conditions?

MTMR9-FITC antibodies can help investigate MTMR9's role in disease through:

• Neurodegenerative disease models:

  • MTMR9 is expressed in brain tissue and may influence neuronal function

  • Compare MTMR9 expression and localization in:

    • Alzheimer's disease models (altered phosphoinositide metabolism)

    • Parkinson's disease models (autophagy dysfunction)

    • ALS models (defective endosomal trafficking)

• Cancer research applications:

  • MTMR9 may function in cell proliferation control

  • Compare expression patterns in:

    • Tumor vs. normal tissue microarrays

    • Cancer cell lines with different metastatic potential

    • Before and after treatment with autophagy or apoptosis modulators

• Metabolic disorder investigations:

  • Examine MTMR9 expression in:

    • Insulin-responsive tissues from diabetic models

    • Liver samples from non-alcoholic fatty liver disease

    • Correlate with autophagy markers and lipid droplet formation

• Tissue-specific expression profiling:

  • Use MTMR9-FITC with tissue-specific markers

  • Quantify expression levels across different cell types

  • Correlate with disease progression markers

• Therapeutic response monitoring:

  • Evaluate changes in MTMR9 expression/localization after drug treatment

  • Use as potential biomarker for therapies targeting autophagy or apoptosis