MTMR9 Antibody, HRP conjugated

What is MTMR9 and why is it significant in myotubularin research?

MTMR9 (Myotubularin-related protein 9) is a catalytically inactive member of the myotubularin family, which consists of 14 proteins. Eight members possess catalytic activity as 3-phosphatases, dephosphorylating phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate, while six members, including MTMR9, are catalytically inactive . MTMR9 is significant because it forms heteromeric complexes with catalytically active myotubularins such as MTMR6 and MTMR8, substantially enhancing their enzymatic activity, stability, and biological functions . MTMR9 contains a glycine residue instead of the conserved cysteine residue in the dsPTPase catalytic loop, rendering it catalytically inactive as a phosphatase .

What detection methods are compatible with MTMR9 Antibody, HRP conjugated?

MTMR9 Antibody, HRP conjugated can be used in multiple detection methods including:

• Western blotting (WB)

• Enzyme-linked immunosorbent assay (ELISA)

• Immunohistochemistry (IHC)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

The horseradish peroxidase (HRP) conjugation allows for direct detection using chemiluminescent or colorimetric substrates without requiring secondary antibodies, simplifying the experimental protocol and potentially reducing background signal . Detection sensitivity can be optimized using enhanced chemiluminescence (ECL) systems, as demonstrated in studies examining MTMR protein interactions .

What are the recommended storage conditions for maintaining MTMR9 Antibody, HRP conjugated activity?

For optimal activity preservation of MTMR9 Antibody, HRP conjugated:

• Store at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles

• For short-term storage and frequent use, store at 4°C for up to one month

• Some formulations contain 50% glycerol in PBS with preservatives such as 0.03% Proclin 300 or 0.02% sodium azide at pH 7.4

Proper aliquoting upon receipt is recommended to minimize freeze-thaw cycles that can significantly reduce antibody activity and binding affinity. Long-term stability studies indicate that antibodies stored according to these recommendations maintain activity for at least one year after shipment .

How should MTMR9 Antibody, HRP conjugated be optimized for Western blot analysis of MTMR9-myotubularin complexes?

When analyzing MTMR9-myotubularin complexes by Western blot:

• Sample preparation:

  • Include both denaturing (reducing) and non-denaturing conditions to detect both monomeric MTMR9 (63 kDa) and heteromeric complexes

  • Consider using crosslinking agents before lysis if studying transient interactions

• Dilution optimization:

  • Start with 1:500-1:2000 dilution range for Western blotting

  • Perform titration experiments if signal-to-noise ratio is suboptimal

• Detection of complexes:

  • MTMR9 appears to form homodimers in addition to heteromeric complexes with MTMR6 and MTMR8

  • When studying MTMR9-MTMR6 complexes, expected molecular weights are approximately 63 kDa for MTMR9 and 72 kDa for MTMR6

• Controls:

  • Include recombinant MTMR9 protein as positive control

  • Use tissues with known MTMR9 expression such as brain tissue

  • RNAi knockdown samples are valuable negative controls

Research has shown that co-expression of MTMR9 with partners like MTMR6 increases protein stability, extending half-life from approximately 40 minutes to 4 hours . This effect should be considered when interpreting western blot band intensities in co-expression experiments.

What strategies can be employed to investigate MTMR9-dependent regulation of active myotubularins using HRP-conjugated antibodies?

To investigate MTMR9-dependent regulation of active myotubularins:

• Co-immunoprecipitation studies:

  • Use anti-FLAG or anti-HA tag antibodies to pull down tagged MTMR9 or partner proteins

  • Detect interactions using HRP-conjugated MTMR9 antibody

  • Reciprocal immunoprecipitation confirms direct interactions

• Enzymatic activity assays:

  • Monitor phosphoinositide phosphatase activity using:

    • In vitro assays measuring release of [³²P]-PO₄ from labeled substrates

    • Cellular assays measuring PtdIns(3)P or PtdIns(3,5)P₂ levels

  • Comparative analysis with and without MTMR9 expression

• Protein stability experiments:

  • Cycloheximide chase assays to measure protein turnover rates

  • Compare half-life of myotubularins with/without MTMR9 co-expression

  • Western blot analysis at multiple time points (0, 2, 4, 6, 8 hours)

• Subcellular localization:

  • Immunofluorescence microscopy to determine co-localization patterns

  • Analysis of MTMR9 impact on localization of active partners

Research has documented that MTMR9 increases MTMR6 enzymatic activity up to 6-fold, and when combined with phosphatidylserine liposomes, activity increases up to 84-fold . Similar studies with MTMR8 showed MTMR9 increased activity 4-fold toward PtdIns(3)P and 1.4-fold toward PtdIns(3,5)P₂ .

How can researchers address potential cross-reactivity issues with MTMR9 Antibody, HRP conjugated?

When addressing cross-reactivity concerns:

• Validation experiments:

  • Perform comparative Western blots using multiple MTMR9 antibodies targeting different epitopes

  • Include knockout/knockdown controls using RNAi (demonstrated to reduce MTMR9 protein by approximately 50%)

  • Test reactivity against recombinant MTMR9 and other myotubularin proteins

• Epitope mapping:

  • Determine the specific region recognized by the antibody

  • Available antibodies target different regions: some target C-terminal (residues 451-549 or 518-549) , while others target middle regions (322-549)

  • Sequence alignment analysis with other myotubularins to identify potential cross-reactive regions

• Blocking peptide experiments:

  • Pre-incubate antibody with excess immunogen peptide

  • Compare signal with and without blocking peptide to confirm specificity

• Species cross-reactivity:

  • Validate reactivity across species if working with non-human models

  • Confirmed reactivity includes human, mouse, and rat for many commercially available antibodies

Cross-reactivity with other myotubularin family members is particularly important to evaluate, as MTMR9 shares sequence homology with other family members and has been incorrectly labeled as MTMR8 in some databases and literature .

What factors affect the sensitivity and specificity of MTMR9 detection using HRP-conjugated antibodies?

Several factors impact detection sensitivity and specificity:

• Buffer composition:

  • Blocking buffer optimization: Testing BSA vs. non-fat dry milk vs. commercial blockers

  • Detergent concentration: 0.1-0.5% Triton X-100 or Tween-20 affects background

  • Salt concentration: 125-150 mM NaCl or potassium acetate optimizes specific binding

• Antibody characteristics:

  • Clonality: Polyclonal antibodies offer higher sensitivity but potentially lower specificity

  • Epitope location: Antibodies targeting different regions may have different accessibility

  • HRP conjugation ratio: Optimal enzyme:antibody ratio for maximum sensitivity

• Tissue/sample preparation:

  • Fixation methods significantly impact epitope availability

  • Protein extraction methods affect native conformations

  • Expression levels vary by tissue (brain tissue shows consistent expression)

• Detection system:

  • Enhanced chemiluminescence (ECL) substrate selection based on expected expression levels

  • Exposure time optimization

  • Digital imaging systems vs. film for quantification purposes

Research protocols typically use antibody dilutions of 1:500-1:2000 for Western blot and 1:50-1:200 for immunohistochemistry and immunofluorescence . Validation studies show observed molecular weight of 60-63 kDa for MTMR9, consistent with its calculated molecular weight of 63 kDa .

How can researchers optimize co-immunoprecipitation experiments to investigate MTMR9 interactions using HRP-conjugated antibodies?

For optimal co-immunoprecipitation results:

• Lysis conditions:

  • Buffer composition: 25 mM Hepes, pH 7.2, 125 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM DTT, 0.4% Triton X-100, and protease inhibitors

  • Gentle lysis methods to preserve protein complexes

  • Temperature control (4°C) throughout the procedure

• Immunoprecipitation strategy:

  • Direct IP using MTMR9 antibody vs. tag-based IP systems

  • Pre-clearing lysates with appropriate control beads

  • Optimizing antibody:lysate ratios and incubation times (overnight at 4°C on a rotator)

• Washing conditions:

  • Number of washes (typically 3-4)

  • Wash buffer stringency affects specificity

  • Bead type selection (protein A/G, magnetic vs. agarose)

• Detection methodologies:

  • Direct detection using MTMR9 Antibody, HRP conjugated

  • Sequential probing for interaction partners

  • Controls: IgG control, input sample (typically 5-10%), non-interacting protein control

Published protocols demonstrate successful co-immunoprecipitation of MTMR9 with both MTMR6 and MTMR8, confirming these direct interactions . Both GST-based pull-down assays and reciprocal co-immunoprecipitation experiments have validated these interactions, showing that complex formation increases protein stability and enzymatic activity of the catalytically active partners .

What is the recommended protocol for using MTMR9 Antibody, HRP conjugated in ELISA applications?

For ELISA applications with MTMR9 Antibody, HRP conjugated:

• Plate preparation:

  • Coat high-binding 96-well plates with capture antibody (if sandwich ELISA) or antigen

  • Optimal coating buffer: Carbonate-bicarbonate buffer pH 9.6

  • Coating concentration: 1-10 μg/ml

  • Incubation: Overnight at 4°C

• Blocking and sample addition:

  • Block with 1-5% BSA or non-fat dry milk in PBS or TBS

  • Sample dilution in blocking buffer with 0.05% Tween-20

  • Incubation: 1-2 hours at room temperature

• Antibody incubation:

  • MTMR9 Antibody, HRP conjugated dilution: 1:1000-1:5000

  • Incubation: 1-2 hours at room temperature

  • Washing: 4-6 times with PBS-T or TBS-T

• Detection and analysis:

  • TMB substrate addition: 100 μl/well

  • Incubation: 10-30 minutes in the dark

  • Stop reaction: 50-100 μl 2N H₂SO₄ or 1N HCl

  • Read absorbance: 450 nm with 570 nm reference

The Mouse MTMR9 ELISA Kit shows a detection range of 0.16-10 ng/mL with a sensitivity of 0.057 ng/mL, providing excellent specificity with minimal cross-reactivity . A typical standard curve should show linearity within the detection range and recovery rates of 85-105% across different matrices including serum (95-105%), EDTA plasma (93-101%), and heparin plasma (85-103%) .

How can MTMR9 Antibody, HRP conjugated be utilized to investigate the cellular functions of MTMR9 in apoptosis regulation?

To investigate MTMR9's role in apoptosis regulation:

• Expression manipulation studies:

  • Overexpression of MTMR9 alone vs. MTMR9 with MTMR6/MTMR8

  • RNA interference targeting MTMR9, MTMR6, or both

  • Analysis of protein levels by Western blot using MTMR9 Antibody, HRP conjugated

• Apoptosis induction and measurement:

  • Treatment with etoposide (100 μM) or other apoptotic stimuli

  • Flow cytometry analysis for apoptotic markers

  • Comparison of cell death between control, MTMR9 knockdown, MTMR6 knockdown, and double knockdown conditions

• Signaling pathway analysis:

  • Phosphoinositide level measurement

  • Downstream effector phosphorylation status

  • Temporal analysis of signaling events

Research has shown that knocking down both MTMR6 and MTMR9 led to increased apoptosis in response to etoposide treatment compared to MTMR6 knockdown alone . Interestingly, MTMR9 knockdown alone also led to significant cell death, suggesting it may interact with multiple members of the myotubularin family involved in apoptosis regulation . When coexpressed, MTMR6 and MTMR9 decreased etoposide-induced apoptosis, indicating a protective function for this complex .

What techniques can be employed to analyze MTMR9's impact on the enzymatic activity of myotubularin phosphatases using HRP-conjugated antibodies?

To analyze MTMR9's impact on myotubularin phosphatase activity:

• In vitro phosphatase assays:

  • Purified proteins (with/without MTMR9)

  • Radiolabeled substrates: PtdIns(3)P and PtdIns(3,5)P₂

  • Quantification of released [³²P]-PO₄

  • Comparison of enzyme kinetics (Km, Vmax)

• Cellular phosphoinositide measurements:

  • Transfection with MTMR9, active myotubularins, or both

  • Antibody-based detection of specific phosphoinositides

  • Quantification of PtdIns(3)P-positive vesicles

  • Analysis of PtdIns(5)P production

• Complex formation and stability:

  • Co-immunoprecipitation using HRP-conjugated antibodies

  • Western blot analysis of protein levels over time

  • Cycloheximide chase experiments

Experimental data demonstrates that MTMR9 increases MTMR6 3-phosphatase activity up to 6-fold . When MTMR6 activity was measured in the presence of phosphatidylserine liposomes (which themselves increased activity 28-fold), the combined effect with MTMR9 resulted in an 84-fold activity increase . Similar studies with MTMR8 showed substrate-specific effects: MTMR9 increased MTMR8 activity 4-fold toward PtdIns(3)P but only 1.4-fold toward PtdIns(3,5)P₂ .

How can researchers investigate the structural determinants of MTMR9 interactions with active myotubularins?

To investigate structural determinants of MTMR9 interactions:

• Domain deletion and mutation studies:

  • Generate truncated or mutated constructs of MTMR9

  • Assess interaction with myotubularin partners by co-immunoprecipitation

  • Evaluate effects on enzymatic activity and protein stability

  • Western blot analysis using MTMR9 Antibody, HRP conjugated

• Structural analysis approaches:

  • Recombinant protein expression and purification

  • Co-crystallization attempts

  • Bioinformatic analysis of conserved interaction domains

  • Examination of the double-helical motif similar to SET interaction domain

• Functional rescue experiments:

  • MTMR9 knockdown followed by rescue with wild-type or mutant constructs

  • Analysis of phosphoinositide levels and cellular phenotypes

  • Quantification of protein complex formation

MTMR9 contains a double-helical motif similar to the SET interaction domain and may function in the control of cell proliferation . Unlike other members of the myotubularin-related protein family, MTMR9 does not contain a functional dual-specificity phosphatase domain and is classified as a pseudophosphatase . This structure allows it to interact with multiple myotubularin family members including MTMR6, MTMR7, and MTMR8, serving as an adapter that enhances their activities .

What are the key differences in experimental approaches when using MTMR9 Antibody, HRP conjugated versus unconjugated primary antibodies?

When comparing HRP-conjugated versus unconjugated MTMR9 antibodies:

Multiple detection formats are available commercially including unconjugated, HRP-conjugated, and other conjugates (FITC, PE, Alexa Fluor) . For co-localization studies of MTMR9 with binding partners, unconjugated primary antibodies from different host species offer advantages for simultaneous detection .

What controls and validation steps are essential when using MTMR9 Antibody, HRP conjugated in research applications?

Essential controls and validation steps include:

• Antibody validation controls:

  • Positive control: Samples with confirmed MTMR9 expression (e.g., brain tissue)

  • Negative control: MTMR9 knockdown or knockout samples

  • Isotype control: Non-specific IgG of the same species and isotype

  • Blocking peptide control: Pre-incubation with immunizing peptide

• Assay-specific controls:

  • For Western blot: Molecular weight markers, loading controls

  • For immunoprecipitation: IgG control, input sample

  • For immunohistochemistry: No primary antibody, isotype control

  • For ELISA: Standard curve, blank wells

• Cross-reactivity assessment:

  • Testing against related myotubularin family proteins

  • Species cross-reactivity confirmation

  • Testing in multiple sample types

• Reproducibility considerations:

  • Batch-to-batch consistency testing

  • Protocol standardization

  • Independent antibody validation using different detection methods

Validation data should include observed molecular weight (60-63 kDa for MTMR9) , expected tissue or cellular distribution patterns, and confirmation of specificity through RNAi experiments which typically show 50% reduction in protein levels with standard RNAi approaches .

How do experimental conditions affect the detection of MTMR9-myotubularin interactions when using HRP-conjugated antibodies?

Experimental conditions affecting detection of MTMR9 interactions:

• Buffer composition effects:

  • Salt concentration: 125-150 mM potassium acetate or NaCl optimal

  • pH: Typically 7.2-7.4 for optimal interaction

  • Detergent type and concentration: 0.4-0.5% Triton X-100 preserves interactions

  • Reducing agents: 1 mM DTT supports protein stability

• Temperature considerations:

  • Complex formation: 4°C for immunoprecipitation studies

  • Antibody incubations: Room temperature vs. 4°C

  • Sample handling: Consistent temperature control throughout

• Time-dependent factors:

  • Incubation duration: Overnight for immunoprecipitation

  • Expression time: 24-48 hours post-transfection for optimal expression

  • Kinetics of complex formation

• Protein expression levels:

  • Overexpression artifacts vs. endogenous levels

  • Ratio of MTMR9 to binding partners

  • Stability enhancement: Co-expression increases protein levels of both partners