MTMR12 Antibody, FITC conjugated

What is MTMR12 and why is it important in research?

MTMR12 (Myotubularin-related protein 12) is a catalytically inactive phosphatase that functions as an adapter for phosphatase myotubularin, regulating its intracellular localization. This protein plays important roles in cellular signaling pathways and has been implicated in various physiological processes. The study of MTMR12 is valuable for understanding phosphatase regulation mechanisms and their implications in cell biology. The MTMR12 protein is encoded by the MTMR12 gene (Gene ID: 54545), and has a UniProt ID of Q9C0I1, providing standardized reference points for research applications .

What are the key specifications of MTMR12 Antibody with FITC conjugation?

The MTMR12 Antibody (FITC) is a rabbit polyclonal antibody conjugated with fluorescein isothiocyanate (FITC) specifically designed for the detection of human MTMR12. The antibody has a purity level exceeding 95% and is purified using Protein G chromatography. It is supplied in liquid form, buffered in 0.01 M PBS (pH 7.4) containing 0.03% Proclin-300 and 50% glycerol. This antibody formulation ensures stability while maintaining reactivity for research applications .

What detection methods are compatible with FITC-conjugated MTMR12 Antibody?

FITC-conjugated MTMR12 Antibody is primarily designed for fluorescence-based detection methods. The FITC fluorophore (excitation maximum: ~495 nm, emission maximum: ~519 nm) makes this antibody particularly suitable for:

• Fluorescence microscopy

• Flow cytometry

• Immunocytochemistry

• Immunofluorescence assays

The direct fluorophore conjugation eliminates the need for secondary antibody incubation steps, simplifying experimental workflows and reducing background in multiple-labeling experiments .

How should optimal dilutions be determined for FITC-conjugated MTMR12 Antibody?

Determining optimal dilutions for FITC-conjugated MTMR12 Antibody requires systematic titration experiments. Begin with the manufacturer's recommended dilution range and perform a dilution series (e.g., 1:50, 1:100, 1:200, 1:500) on your specific sample type. Evaluate signal-to-noise ratio at each dilution to identify the concentration that provides maximum specific signal with minimal background. Different applications may require different optimal dilutions - immunofluorescence typically requires more concentrated antibody than flow cytometry. Always include appropriate positive and negative controls to establish specificity. Document optimal conditions once determined for reproducibility in future experiments .

What sample preparation protocols are recommended for MTMR12 Antibody (FITC) immunostaining?

For optimal results with MTMR12 Antibody (FITC) immunostaining, follow these methodological steps:

• Cell fixation: Fix cells with 4% paraformaldehyde (10-15 minutes at room temperature) to preserve cellular architecture while maintaining epitope accessibility.

• Permeabilization: Use 0.1-0.5% Triton X-100 in PBS (5-10 minutes) to allow antibody access to intracellular targets.

• Blocking: Incubate samples with 1-5% BSA or normal serum in PBS (30-60 minutes) to minimize non-specific binding.

• Antibody dilution: Prepare the FITC-conjugated MTMR12 antibody in blocking buffer at the optimized dilution.

• Incubation: Apply diluted antibody to samples and incubate (1-2 hours at room temperature or overnight at 4°C) in a humidified, dark chamber to prevent photobleaching.

• Washing: Perform 3-5 thorough washes with PBS to remove unbound antibody.

• Counterstaining: Apply nuclear counterstain if desired (e.g., DAPI).

• Mounting: Mount with anti-fade medium to preserve fluorescence signal during imaging .

How should MTMR12 Antibody (FITC) be stored to maintain optimal activity?

To maintain optimal activity of MTMR12 Antibody (FITC), follow these critical storage protocols:

• Store the antibody at -20°C in the dark to prevent photobleaching of the FITC fluorophore.

• Aliquot the antibody upon first thawing to minimize freeze-thaw cycles, as each cycle can reduce activity.

• When handling the antibody, maintain cold chain conditions and avoid extended exposure to room temperature.

• Avoid repeated freeze-thaw cycles; each cycle potentially reduces antibody activity by 10-15%.

• When removing from storage, thaw the antibody completely and mix gently by inversion (avoid vortexing).

• Protect from prolonged light exposure at all times to prevent photobleaching of the FITC conjugate.

• If used frequently, a small working aliquot can be maintained at 4°C for up to two weeks.

This storage regimen maximizes antibody shelf life (typically stable for one year from shipment when properly stored) .

How does MTMR12 interact with other myotubularin family members in cellular contexts?

MTMR12 functions as a critical adapter protein within the myotubularin family interaction network. While direct data on MTMR12 interactions is limited in the provided search results, we can draw parallels from related MTMR protein interactions. Similar to how MTMR2 interacts with MTMR5, MTMR12 likely forms specific protein-protein interactions with catalytically active myotubularin family members.

These interactions typically involve:

• Coiled-coil domain-mediated binding (as seen between MTMR2 and MTMR5)

• Regulation of phosphatase activity through allosteric mechanisms

• Control of subcellular localization and trafficking

• Formation of functional protein complexes

The specificity of these interactions is crucial, as demonstrated by the fact that MTMR5 interacts with MTMR2 but not MTM1, suggesting highly selective binding mechanisms within this protein family .

What are the methodological considerations for multiplexing MTMR12 Antibody (FITC) with other fluorescent markers?

When designing multiplex fluorescence experiments including MTMR12 Antibody (FITC), consider these methodological factors:

• Spectral compatibility: FITC has excitation/emission peaks at approximately 495/519 nm. Select complementary fluorophores with minimal spectral overlap such as:

  • DAPI for nuclei (Ex/Em: 358/461 nm)

  • Cy3/TRITC for additional targets (Ex/Em: 550/570 nm)

  • Cy5/APC for additional targets (Ex/Em: 650/670 nm)

• Sequential staining protocol:

  • Begin with the weakest signal antibody, typically ending with FITC-conjugated antibodies

  • Include appropriate blocking steps between different antibodies

  • Use species-specific secondary antibodies for unconjugated primaries

• Cross-reactivity prevention:

  • Thoroughly test each antibody individually before multiplexing

  • Use antibodies raised in different host species when possible

  • Consider using F(ab) fragments to reduce non-specific binding

• Controls:

  • Include single-color controls for compensation settings

  • Prepare fluorescence-minus-one (FMO) controls

  • Include isotype controls for each conjugated antibody

• Imaging considerations:

  • Acquire channels sequentially to minimize bleed-through

  • Optimize exposure settings for each fluorophore

  • Apply appropriate spectral unmixing algorithms during analysis .

How does the subcellular localization pattern of MTMR12 compare with other myotubularin family members?

The subcellular localization pattern of MTMR12 shows distinct characteristics compared to other myotubularin family members. While specific MTMR12 localization data is not fully detailed in the search results, we can draw important comparisons based on related family members:

• Regulatory mechanism: MTMR12, as a catalytically inactive member, likely functions as an adapter protein that regulates the localization of active myotubularins. This parallels how MTMR5 regulates MTMR2 localization.

• Coiled-coil domain importance: The coiled-coil domain appears critical for protein-protein interactions within this family, as demonstrated by MTMR2's inability to interact with MTMR5 when this domain is deleted. MTMR12 contains similar structural domains that likely mediate its interactions and subsequent localization patterns.

• Membrane association dynamics: MTMR2 shows dynamic re-localization to membranes under hypo-osmotic conditions. MTMR12 may exhibit similar conditional membrane association patterns dependent on cellular stress or signaling events.

• Dimerization influence: The dimerization state of myotubularins affects their localization. MTMR2 exists as a homodimer while MTM1 is predominantly monomeric. MTMR12's oligomerization state would similarly influence its distribution pattern.

When conducting immunofluorescence studies with MTMR12 Antibody (FITC), researchers should pay particular attention to these comparative localization patterns to understand the functional significance of MTMR12 in different cellular compartments .

What are common causes for weak or absent signal when using MTMR12 Antibody (FITC)?

When encountering weak or absent signals with MTMR12 Antibody (FITC), systematically evaluate these potential causes:

• Antibody degradation:

  • FITC is sensitive to photobleaching; verify storage conditions were maintained

  • Check for extended exposure to light or improper freezing

  • Verify absence of bacterial contamination in antibody solution

• Sample-related issues:

  • Insufficient target protein expression in sample

  • Over-fixation causing epitope masking or destruction

  • Inadequate permeabilization preventing antibody access

  • MTMR12 expression varies by cell type; HEK-293, K-562, and HeLa cells show detectable expression

• Protocol optimization:

  • Insufficient antibody concentration (try 2-5× higher concentration)

  • Inadequate incubation time or temperature

  • Improper blocking leading to high background masking signal

  • pH conditions outside optimal range affecting FITC fluorescence (optimal pH: 7.2-8.0)

• Technical factors:

  • Incorrect filter sets on imaging equipment

  • Suboptimal microscope settings (exposure time, gain)

  • Photobleaching during extended imaging sessions

• Validation approach:

  • Test antibody on positive control samples (K-562 or HEK-293 cells)

  • Perform parallel experiments with alternate detection methods

  • Consider using signal amplification methods for low-abundance targets

How can researchers validate the specificity of MTMR12 Antibody (FITC) in their experimental system?

To rigorously validate MTMR12 Antibody (FITC) specificity in your experimental system, implement this comprehensive validation strategy:

• Positive controls:

  • Use cell lines known to express MTMR12 (K-562, HEK-293, HeLa cells)

  • Include recombinant MTMR12 protein as a standard

  • Compare staining pattern with published literature

• Negative controls:

  • Use isotype-matched control antibody conjugated to FITC

  • Include cells with confirmed absence or knockdown of MTMR12

  • Perform blocking peptide experiments if competing peptide is available

• Cross-validation approaches:

  • Compare results with alternative MTMR12 antibodies from different sources

  • Correlate immunofluorescence patterns with Western blot results

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

• Genetic verification:

  • Test antibody in MTMR12 knockdown/knockout models

  • Perform siRNA experiments with graduated reduction of target

  • Correlate protein detection with mRNA expression levels

• Peptide competition assay:

  • Pre-incubate antibody with excess immunizing peptide

  • Compare staining with and without peptide competition

  • Specific signal should be significantly reduced or eliminated

• Microscopy controls:

  • Image at multiple exposure settings to ensure signal is not autofluorescence

  • Use spectral imaging to confirm emission profile matches FITC

  • Perform Z-stack imaging to confirm subcellular localization pattern is consistent

What experimental approaches can resolve contradictory results when studying MTMR12 interactions?

When faced with contradictory results in MTMR12 interaction studies, implement these methodological approaches to resolve discrepancies:

• Multiple detection techniques:

  • Compare results across complementary methods:

    • Co-immunoprecipitation for endogenous protein interactions

    • Proximity ligation assay for in situ interaction visualization

    • FRET/BRET for real-time interaction dynamics

    • GST pull-down assays for direct binding assessment

  • Each technique has distinct strengths and limitations that can explain contradictory findings

• Domain mapping analysis:

  • Create and test domain deletion mutants to identify critical interaction regions

  • Similar to how MTMR2's coiled-coil domain was identified as essential for MTMR5 interaction

  • Point mutations can provide finer resolution of binding interfaces

• Cell type and condition considerations:

  • Test interactions across multiple cell types

  • Evaluate effects of cell confluence, serum conditions, and stress states

  • Some interactions may be cell-type specific or condition-dependent

• Post-translational modification analysis:

  • Phosphorylation state can dramatically affect protein interactions

  • Use phosphatase inhibitors or phosphomimetic mutations

  • Analyze interactions under conditions affecting PTM status

• Quantitative binding assessment:

  • Employ surface plasmon resonance or bio-layer interferometry

  • Determine binding affinities under various conditions

  • Weak interactions may be detected by some methods but not others

• Computational validation:

  • Use protein modeling to predict interaction interfaces

  • Molecular dynamics simulations can reveal interaction mechanisms

  • Phylogenetic analysis can identify conserved interaction motifs

How does MTMR12 expression and localization change under different cellular stress conditions?

MTMR12 expression and localization demonstrate dynamic responses to various cellular stress conditions, though specific data on MTMR12 is limited in the search results. Based on patterns observed with related family members such as MTMR2, we can infer several important stress-responsive characteristics:

• Osmotic stress response:

  • Similar to MTMR2, which relocates to membranes under hypo-osmotic conditions, MTMR12 likely undergoes stress-induced subcellular redistribution

  • This relocation may reflect a role in membrane remodeling or phosphoinositide signaling during osmotic adaptation

• Oxidative stress effects:

  • Reactive oxygen species may alter MTMR12 function through post-translational modifications

  • Oxidation of critical cysteine residues could affect protein-protein interactions or regulatory functions

• Nutrient deprivation responses:

  • Autophagy induction during starvation may involve MTMR12 regulation

  • As an adapter for active phosphatases, MTMR12 could modulate phosphoinositide pools during autophagic processes

• Heat shock conditions:

  • Temperature stress may affect protein folding and interaction capacity

  • Chaperone association with MTMR12 under heat shock could be evaluated using co-immunoprecipitation

• Experimental approaches:

  • Time-course immunofluorescence using MTMR12 Antibody (FITC) under various stress conditions

  • Subcellular fractionation followed by Western blotting to quantify redistribution

  • Live-cell imaging with fluorescently-tagged MTMR12 to monitor dynamic responses

  • Comparison with other myotubularin family members to identify common stress response patterns

What are the methodological considerations for studying MTMR12 in animal disease models?

When designing studies of MTMR12 in animal disease models, researchers should address these methodological considerations:

• Model selection rationale:

  • Choose models where phosphoinositide signaling is implicated

  • Consider genetic models with MTMR12 knockout/knockdown

  • Mouse models are well-validated, with confirmed reactivity to available MTMR12 antibodies

  • Rat models offer advantages for certain neurological studies

• Tissue-specific expression analysis:

  • MTMR12 shows varied expression across tissues, with notable presence in:

    • Brain tissue (validated in mouse brain)

    • Lung tissue (validated in mouse lung)

  • Optimize immunohistochemistry protocols for each tissue type

• Cross-species antibody validation:

  • Confirm MTMR12 Antibody (FITC) reactivity in your animal model

  • Available antibodies show reactivity with human, mouse, and rat samples

  • Western blot validation should precede immunohistochemical applications

• Interaction partners assessment:

  • Investigate conserved interactions across species

  • Co-immunoprecipitation can identify species-specific interaction differences

  • Consider proximity ligation assays for in situ interaction visualization

• Developmental timing considerations:

  • Expression patterns may vary during development

  • Age-matched controls are essential for meaningful comparisons

  • Longitudinal studies may reveal temporal regulation patterns

• Quantification approaches:

  • Standardize image acquisition settings for immunofluorescence

  • Use appropriate internal controls for expression normalization

  • Consider automated analysis algorithms to reduce bias in localization studies

What is the current understanding of MTMR12's role in disease pathology compared to other myotubularin family members?

The current understanding of MTMR12's role in disease pathology, compared to other myotubularin family members, reveals distinct contributions to human disorders:

• Comparative disease associations:

  • MTMR2 mutations cause Charcot-Marie-Tooth disease type 4B1 (CMT4B1), a demyelinating neuropathy

  • MTM1 mutations lead to X-linked myotubular myopathy

  • MTMR12's specific disease associations are less characterized but may involve modulation of these related disorders through its adapter function

• Functional distinctions:

  • MTMR12 is catalytically inactive, functioning primarily as an adapter protein

  • This contrasts with catalytically active members like MTM1 and MTMR2

  • MTMR12 likely exerts its disease influence through regulation of active phosphatases

• Regulatory mechanisms:

  • The interaction of MTMR12 with active myotubularins suggests its mutations could dysregulate multiple phosphatase activities

  • This regulatory role may create broader phenotypic effects than mutations in single active phosphatases

• Experimental evidence:

  • MTMR12's role as an adapter for phosphatase myotubularin parallels MTMR5's regulation of MTMR2

  • The influence on intracellular localization suggests MTMR12 controls the spatial aspects of phosphoinositide signaling

• Mechanistic hypotheses:

  • MTMR12 dysfunction may disrupt phosphoinositide balance

  • This disruption potentially affects membrane trafficking, a common pathway in myotubularin-related diseases

  • Vesicular trafficking abnormalities in Schwann cells (as seen with MTMR2) could be influenced by MTMR12 status

• Research implications:

  • MTMR12 represents a potential therapeutic target for modulating active myotubularin function

  • Studying MTMR12 interactions may reveal common mechanisms underlying multiple myotubularin-related disorders

What are the optimal parameters for using MTMR12 Antibody (FITC) in flow cytometry experiments?

For optimal flow cytometry experiments using MTMR12 Antibody (FITC), implement these technical parameters:

• Sample preparation protocol:

  • Harvest cells in mid-log growth phase for consistent expression

  • Fix with 2-4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 0.1% saponin or 0.1% Triton X-100 for intracellular staining

  • Maintain cell concentration at 1×10^6 cells/mL for consistent results

• Antibody titration:

  • Perform a dilution series (1:50, 1:100, 1:200, 1:500)

  • Plot staining index (ratio of positive signal to background) for each dilution

  • Select concentration with highest staining index, typically starting at 1:100

  • Include unstained and isotype controls for accurate gating

• Staining conditions:

  • Incubate cells with antibody for 30-45 minutes at 4°C in the dark

  • Use staining buffer containing 1-2% FBS or BSA to reduce non-specific binding

  • Include 0.1% sodium azide to prevent internalization during staining

  • Wash cells 2-3 times with excess buffer after staining

• Instrument settings:

  • Excite FITC with 488 nm laser

  • Collect emission using 530/30 nm bandpass filter

  • Optimize voltage settings using unstained cells and single-color controls

  • Run positive control samples (K-562 or HEK-293 cells) to validate settings

• Data analysis considerations:

  • Gate on single cells using FSC-A vs. FSC-H

  • Exclude dead cells using appropriate viability dye

  • Set positive/negative boundaries using isotype control

  • Consider mean fluorescence intensity for quantitative comparisons

How can researchers optimize double immunostaining protocols involving MTMR12 Antibody (FITC) and other myotubularin family antibodies?

To optimize double immunostaining protocols with MTMR12 Antibody (FITC) and other myotubularin family antibodies, follow this methodological framework:

• Antibody selection strategy:

  • Choose unconjugated antibodies for other myotubularin family members

  • Select antibodies raised in different host species than MTMR12 Antibody (FITC)

  • For example, use mouse monoclonal antibodies for MTMR2 detection alongside rabbit-derived MTMR12 Antibody (FITC)

  • Verify specificity of each antibody individually before attempting co-staining

• Sequential staining approach:

  • Apply unconjugated primary antibody first

  • Add species-specific secondary antibody with a compatible fluorophore (e.g., Cy3, Cy5)

  • Block any remaining binding sites with excess IgG from the same species

  • Apply FITC-conjugated MTMR12 antibody last to minimize cross-reactivity

• Optimization parameters:

  • Titrate each antibody separately to determine optimal concentration

  • Test different incubation times and temperatures for each step

  • Evaluate various blocking reagents (BSA, normal serum, commercial blockers)

  • Compare different fixation methods for optimal epitope preservation

• Critical controls:

  • Single antibody controls to assess bleed-through

  • Secondary-only controls to evaluate non-specific binding

  • Peptide competition controls to confirm specificity

  • Cells lacking one or both targets as negative controls

• Technical considerations:

  • Use multiband filter sets or sequential scanning for confocal microscopy

  • Apply spectral unmixing algorithms if fluorophore emission overlaps

  • Consider signal amplification for low-abundance targets

  • Document all parameters for reproducibility

What quantitative analysis methods are recommended for MTMR12 localization studies using immunofluorescence?

For rigorous quantitative analysis of MTMR12 localization in immunofluorescence studies, implement these methodological approaches:

• Colocalization analysis methods:

  • Pearson's correlation coefficient: Measures linear correlation between MTMR12 and organelle markers

  • Manders' overlap coefficient: Quantifies proportional overlap independent of signal intensity

  • Li's intensity correlation analysis: Determines whether signals vary dependently

  • Object-based colocalization: Counts objects positive for both MTMR12 and reference marker

• Subcellular distribution quantification:

  • Line profile analysis: Plot fluorescence intensity across cellular regions

  • Radial profile analysis: Measure intensity distribution from nucleus to periphery

  • Cellular compartment segmentation: Define regions (nucleus, cytoplasm, membrane) and measure relative MTMR12 distribution

  • Distance mapping: Calculate distances between MTMR12 puncta and organelle markers

• Dynamics assessment:

  • FRAP (Fluorescence Recovery After Photobleaching): Measure protein mobility in different compartments

  • Time-lapse imaging: Track MTMR12 redistribution following stimuli

  • Pulse-chase labeling: Monitor protein trafficking pathways

• Statistical approaches:

  • Use multiple biological replicates (minimum n=3)

  • Analyze adequate cell numbers per condition (typically >30 cells)

  • Apply appropriate statistical tests (ANOVA with post-hoc analysis for multiple comparisons)

  • Report effect sizes alongside p-values

• Software recommendations:

  • ImageJ/Fiji with JACoP plugin: Comprehensive colocalization analysis

  • CellProfiler: Automated detection and measurement of subcellular patterns

  • Imaris: 3D visualization and quantification

  • Custom MATLAB or Python scripts: For specialized analyses

• Standardization practices:

  • Use identical acquisition settings across all samples

  • Apply consistent thresholding methods

  • Include internal reference standards

  • Document all image processing steps for reproducibility