MTMR3 Antibody, FITC conjugated

What is MTMR3 and why is it important for research?

MTMR3 (Myotubularin Related Protein 3) functions as a phosphoinositide phosphatase that hydrolyzes phosphatidylinositol 3-phosphate and phosphatidylinositol 3,5-bisphosphate. It plays critical roles in membrane trafficking and cytoskeletal dynamics, which are essential for maintaining cellular integrity and function. Additionally, MTMR3 may dephosphorylate proteins containing phosphorylated serine, threonine, and tyrosine residues. The significance of MTMR3 extends to neurobiology, as mutations in related proteins can lead to severe neurological disorders such as Charcot-Marie-Tooth disease, highlighting its importance in both physiological and pathological processes .

What are the key specifications of MTMR3 Antibody, FITC conjugated?

The MTMR3 Antibody, FITC conjugated, is a rabbit polyclonal antibody targeting amino acids 652-899 of human MTMR3. It has been purified to >95% purity using Protein G chromatography and is supplied in liquid form. The buffer contains 0.03% Proclin 300 as a preservative, 50% glycerol, and 0.01M PBS at pH 7.4. The immunogen used for production is a recombinant human MTMR3 protein fragment (amino acids 652-899). The antibody is of IgG isotype and demonstrates specific reactivity with human MTMR3 .

What is the significance of the FITC conjugation?

The FITC (Fluorescein Isothiocyanate) conjugation enables direct visualization of MTMR3 in various fluorescence-based applications without requiring secondary antibodies. This conjugation emits green fluorescence when excited at the appropriate wavelength, allowing for direct detection in immunofluorescence microscopy, flow cytometry, and other fluorescence-based techniques. The FITC conjugation simplifies experimental protocols by eliminating the need for secondary detection systems, reducing background signal, and enabling multicolor staining when used with antibodies conjugated to spectrally distinct fluorophores .

What are the validated applications for MTMR3 Antibody, FITC conjugated?

While specific applications require validation by end-users, the MTMR3 Antibody, FITC conjugated, is potentially suitable for immunofluorescence (IF), immunohistochemistry (IHC), enzyme-linked immunosorbent assay (ELISA), and flow cytometry (FCM). The antibody's applications should be determined empirically for each experimental system, and optimal dilutions should be established through titration experiments. Unlike some other MTMR3 antibodies that have been validated for Western blotting, the specific FITC-conjugated version primarily targets applications leveraging fluorescence detection capabilities .

How can I optimize immunofluorescence protocols with this antibody?

For optimal immunofluorescence results with MTMR3 Antibody, FITC conjugated, implement the following methodological approach:

• Fix cells using 4% paraformaldehyde (10-15 minutes) followed by permeabilization with 0.1% Triton X-100 (5-10 minutes)

• Block with 1-5% BSA or normal serum for 30-60 minutes to reduce non-specific binding

• Determine optimal antibody concentration through titration (typically starting at 1:100-1:500)

• Incubate samples with antibody solution (2-12 hours, depending on temperature)

• Perform stringent washing steps to minimize background

• Mount using anti-fade mounting medium containing a nuclear counterstain

• Protect from light during all steps after antibody addition to prevent photobleaching of the FITC conjugate

• Include appropriate negative controls (isotype control) and positive controls (samples known to express MTMR3)

What cell types and tissues are most appropriate for studying MTMR3 expression?

Based on its biological functions, MTMR3 expression and activity can be studied in various cell types involved in membrane trafficking, autophagy, and lipid signaling. Neuronal cells are particularly relevant due to MTMR3's association with neurological disorders through related family members. Additionally, cells with active endosomal-lysosomal systems would be appropriate for investigating MTMR3's role in membrane dynamics. While the antibody specifically reacts with human MTMR3, researchers should verify expression patterns in their specific cell types of interest before conducting extensive studies .

How do I design experiments to investigate MTMR3's phosphatase activity using this antibody?

To investigate MTMR3's phosphatase activity while utilizing this FITC-conjugated antibody, implement a multi-method experimental design:

• Use the antibody for localization studies to determine MTMR3 subcellular distribution in relation to its phosphoinositide substrates

• Combine with phosphoinositide sensors (e.g., PH domains fused to spectrally distinct fluorophores) to visualize substrate-enzyme co-localization

• Perform co-localization studies with markers for endosomal compartments (e.g., Rab5, Rab7) to analyze MTMR3 activity sites

• Design functional assays combining visualization (using the FITC-antibody) with biochemical measurements of phosphoinositide levels

• For advanced studies, combine with pharmacological inhibitors of phosphoinositide metabolism to dissect pathway components

• Consider complementary approaches such as knockdown/knockout followed by rescue experiments to confirm specificity of observed effects

What controls should be included when using MTMR3 Antibody, FITC conjugated?

A robust experimental design with MTMR3 Antibody, FITC conjugated, requires comprehensive controls:

• Isotype control: Rabbit IgG-FITC at the same concentration to assess non-specific binding

• Negative control samples: Cells/tissues with confirmed low or no MTMR3 expression

• Positive control samples: Cells/tissues with confirmed MTMR3 expression

• Antibody titration controls: Serial dilutions to determine optimal concentration

• Absorption controls: Pre-incubation of antibody with immunizing peptide to confirm specificity

• Secondary-only controls: For assessing background in multi-labeling experiments

• FITC autofluorescence control: Unstained samples to establish baseline fluorescence

• siRNA/shRNA knockdown controls: To validate antibody specificity and signal reduction following MTMR3 depletion

How can I assess MTMR3 interactions with other MTMR family members using this antibody?

To investigate MTMR3 interactions with other family members such as MTMR4:

• Design co-immunoprecipitation experiments using non-FITC conjugated MTMR3 antibodies followed by detection of interacting partners

• Perform dual immunofluorescence studies combining the MTMR3-FITC antibody with antibodies against potential interacting partners labeled with spectrally distinct fluorophores

• Utilize proximity ligation assays (PLA) to detect protein-protein interactions in situ

• Implement FRET (Fluorescence Resonance Energy Transfer) approaches using MTMR3-FITC as donor and another fluorophore-conjugated antibody as acceptor

• Combine with genetic approaches (overexpression, knockdown) to manipulate expression levels of potential interacting partners

• Consider using this antibody in live-cell imaging to track dynamic interactions if cell permeabilization protocols can be optimized

How should I handle and store MTMR3 Antibody, FITC conjugated for optimal performance?

To maintain antibody integrity and performance:

• Upon receipt, aliquot the antibody to minimize freeze-thaw cycles

• Store at -20°C or -80°C as recommended by the manufacturer

• Protect from light at all times due to FITC photosensitivity

• Avoid repeated freeze-thaw cycles as they may lead to denaturation and loss of activity

• When thawing, place on ice and protect from light

• Centrifuge briefly before opening to collect liquid at the bottom of the tube

• Consider adding additional preservatives if diluting the antibody for longer-term storage

• Monitor the performance of older aliquots against fresh aliquots to assess stability

What are common issues in immunofluorescence using FITC-conjugated antibodies and how can they be resolved?

Common technical challenges with FITC-conjugated antibodies include:

• High background signal

  • Solution: Increase blocking time/concentration, optimize antibody dilution, include additional washing steps

• Photobleaching

  • Solution: Minimize exposure to light, use anti-fade mounting media, capture images promptly

• Autofluorescence

  • Solution: Include unstained controls, use spectral unmixing, consider tissue autofluorescence quenchers

• Low signal intensity

  • Solution: Optimize fixation conditions, increase antibody concentration, enhance antigen retrieval

• Non-specific binding

  • Solution: Use more stringent blocking, optimize antibody concentration, include isotype controls

• Signal bleed-through in multi-labeling experiments

  • Solution: Use sequential scanning, adjust acquisition parameters, implement spectral unmixing

How can I validate the specificity of MTMR3 staining with this antibody?

To confirm the specificity of MTMR3 detection:

• Perform siRNA/shRNA knockdown of MTMR3 and verify signal reduction

• Use CRISPR/Cas9 knockout models as negative controls

• Conduct peptide competition assays by pre-incubating the antibody with excess immunizing peptide

• Compare staining patterns with alternative MTMR3 antibodies recognizing different epitopes

• Correlate protein detection with mRNA expression using complementary techniques like RT-PCR or RNA-seq

• Verify subcellular localization against known MTMR3 distribution patterns

• Perform Western blotting with cell lysates to confirm antibody recognizes a protein of the expected molecular weight

• Utilize tissue samples with established MTMR3 expression patterns as anatomical controls

How can MTMR3 Antibody, FITC conjugated be used to study autophagy regulation?

MTMR3 has been implicated in autophagy regulation through its phosphoinositide phosphatase activity. To investigate this role:

• Implement dual labeling with autophagy markers (LC3, p62, WIPI) to assess co-localization during autophagy induction and inhibition

• Combine MTMR3 detection with live-cell imaging of autophagic flux using tandem fluorescent reporters

• Compare MTMR3 localization patterns under normal conditions versus starvation or drug-induced autophagy

• Design experiments to visualize MTMR3 in relation to phosphatidylinositol 3-phosphate pools on autophagic membranes

• Use the antibody in cells with manipulated autophagy pathways (e.g., ATG gene knockouts) to place MTMR3 within the autophagy regulatory network

• Develop quantitative image analysis workflows to measure changes in MTMR3 distribution during autophagy modulation

What approaches can reveal the role of MTMR3 in neurodegenerative diseases?

To investigate MTMR3's potential involvement in neurodegeneration:

• Analyze MTMR3 expression and localization in neuronal models of disease using the FITC-conjugated antibody

• Compare MTMR3 distribution in patient-derived versus healthy control neurons or tissues

• Examine co-localization with disease-associated proteins (e.g., α-synuclein, tau, huntingtin)

• Investigate MTMR3 expression in relation to endosomal-lysosomal dysfunction markers

• Design longitudinal studies to track MTMR3 changes during disease progression

• Combine with functional assays measuring phosphoinositide metabolism in disease models

• Implement high-content screening approaches to identify modulators of MTMR3 activity or expression that affect disease phenotypes

How can I use this antibody in conjunction with super-resolution microscopy?

For super-resolution microscopy applications:

• Verify that the FITC conjugate is compatible with your specific super-resolution technique (STED, PALM, STORM, SIM)

• For STORM/PALM: Consider photoconversion properties of FITC and optimize imaging buffers accordingly

• For STED: Ensure laser lines and filter sets are optimized for FITC excitation and emission

• Implement rigorous controls to distinguish specific signal from background

• Optimize fixation protocols to preserve nanoscale structure while maintaining epitope accessibility

• Consider dual-labeling with spectrally distinct fluorophores for colocalization studies at nanoscale resolution

• Adjust antibody concentration to achieve optimal labeling density for your chosen super-resolution method

• For quantitative analyses, implement appropriate drift correction and calibration procedures