MTMR12 Antibody, Biotin conjugated

What is MTMR12 and why is it significant in research?

MTMR12 (Myotubularin Related Protein 12) is a member of the myotubularin family of phosphatases, playing critical roles in cellular signaling pathways. The protein functions as a scaffolding protein that interacts with active myotubularins, especially MTM1, to regulate phosphoinositide metabolism. MTMR12 has demonstrated significance in both normal cellular function and disease states, particularly in neuromuscular disorders. The protein has a calculated molecular weight of approximately 86 kDa, though it often appears around 73 kDa in experimental conditions, suggesting potential post-translational modifications or alternative splicing that affect its migration pattern in gel electrophoresis . MTMR12 is widely expressed across multiple species, including humans, mice, rats, and various other mammals, making it valuable for comparative studies .

What is the principle behind biotin conjugation of antibodies?

Biotin conjugation involves the covalent attachment of biotin molecules to antibodies, typically ranging from 3-6 biotin molecules per antibody structure . This modification leverages the extremely high affinity between biotin and streptavidin/avidin to create a powerful detection system. The principle works through a two-step process: first, the biotin-conjugated primary antibody binds to the target antigen (in this case, MTMR12); second, streptavidin or avidin conjugated to a reporter molecule (fluorophore, enzyme, etc.) binds to the biotin with exceptional specificity and strength. This amplifies detection sensitivity significantly compared to direct conjugation methods. The biotinylation process is carefully controlled during manufacturing to maintain optimal antibody functionality while providing sufficient biotin molecules for detection purposes .

What are the optimal applications for biotin-conjugated MTMR12 antibodies?

Biotin-conjugated MTMR12 antibodies excel in multiple experimental contexts based on the data from similar conjugated antibodies. The most effective applications include:

For optimal results in flow cytometry applications, biotin-conjugated antibodies should be titrated specifically for your experimental system, as sensitivity can vary based on cell types and instruments . When using biotin-conjugated MTMR12 antibodies for immunoprecipitation, the high affinity purification method employed in antibody preparation ensures specific targeting of MTMR12 protein complexes while minimizing background interactions .

How does the specificity of MTMR12 antibodies vary across species?

The cross-reactivity profile of MTMR12 antibodies demonstrates considerable conservation across species, though with important variations in epitope recognition. Based on the available data:

When selecting an MTMR12 antibody for cross-species studies, researchers should consider that antibodies targeting the middle region (such as ABIN2786613) or C-terminus (such as ABIN7466762) show different cross-reactivity profiles . For studies requiring detection across multiple species, antibodies raised against highly conserved epitopes in the middle region generally provide more consistent results .

What are the recommended sample preparation protocols for MTMR12 detection?

Optimal sample preparation for MTMR12 detection varies by application but follows these essential principles:

For Western Blotting:

• Lyse cells in RIPA buffer supplemented with protease inhibitors.

• Denature samples at 95°C for 5 minutes in reducing sample buffer.

• Load 20-50 μg of total protein per lane.

• Use a 10% SDS-PAGE gel for optimal resolution around the 73-86 kDa range where MTMR12 appears .

For Immunoprecipitation:

• Use 0.5-4.0 μg of antibody for every 1.0-3.0 mg of total protein lysate.

• Pre-clear lysates with protein A/G beads before adding the antibody.

• Incubate with MTMR12 antibody overnight at 4°C with gentle rotation.

• Capture complexes with streptavidin-coated beads (for biotin-conjugated antibodies) .

For Immunohistochemistry:

• For paraffin sections: perform antigen retrieval using citrate buffer (pH 6.0).

• For frozen sections: fix in cold acetone for 10 minutes.

• Block endogenous biotin using a commercial biotin blocking kit.

• Block endogenous peroxidases with 0.3% H₂O₂ if using HRP detection systems .

The detection of MTMR12 can be challenging due to its specific expression patterns and potential post-translational modifications. Validation with positive controls is essential, with K-562 cells, HEK-293 cells, and mouse brain tissue serving as reliable positive controls based on experimental validation .

How should dilution factors be adjusted for different applications?

Dilution optimization is critical for achieving the ideal balance between specific signal and background noise:

The optimal dilution is sample-dependent and should be determined empirically for each experimental system . For biotin-conjugated antibodies specifically, dilutions may need to be adjusted to account for the degree of biotinylation, which can affect antibody binding efficiency. Additionally, when working with tissue samples, background from endogenous biotin should be considered, potentially requiring more stringent blocking steps and higher antibody dilutions .

How can high background issues be resolved when using biotin-conjugated MTMR12 antibodies?

High background is a common challenge with biotin-conjugated antibodies due to endogenous biotin in biological samples. Implement these methodological solutions:

• Endogenous Biotin Blocking: Prior to primary antibody application, block endogenous biotin using commercially available biotin/avidin blocking kits. This step is particularly crucial for tissues rich in endogenous biotin (liver, kidney, brain).

• Antibody Concentration Optimization: Excessive antibody concentrations significantly contribute to background. Perform sequential dilutions from 1:100 to 1:3000 to identify the optimal concentration that balances signal strength with background reduction .

• Buffer Modifications: For Western blotting, add 0.1-0.5% Tween-20 to wash buffers and include 5% BSA rather than milk for blocking, as milk contains endogenous biotin. For immunohistochemistry, include 0.1% Triton X-100 in wash buffers to reduce non-specific membrane associations.

• Detection System Dilution: Often overlooked, the streptavidin conjugate concentration should be titrated independently of the primary antibody. Begin with a 1:200 dilution of streptavidin-HRP and adjust as needed.

• Sample-Specific Considerations: Different MTMR12 validation data show variable background patterns across sample types. For example, K-562 cells demonstrate cleaner results than liver tissue samples, where endogenous biotin levels are higher .

What controls are essential when validating biotin-conjugated MTMR12 antibody experiments?

A comprehensive validation strategy requires multiple control types:

For flow cytometry applications specifically, include a fluorescence-minus-one (FMO) control to accurately set gating parameters when using biotin-conjugated MTMR12 antibodies in multicolor panels . Peptide competition assays are particularly valuable for confirming specificity, especially when using antibodies directed against middle regions (like ABIN2786613) or C-terminal epitopes (like ABIN7466762) of MTMR12 .

How can biotin-conjugated MTMR12 antibodies be utilized for multi-parameter analysis?

Biotin-conjugated MTMR12 antibodies offer exceptional flexibility for multi-parameter studies through several advanced methodological approaches:

• Sequential Immunodetection: For co-localization studies, biotin-conjugated MTMR12 antibodies can be paired with directly-labeled antibodies against interacting partners. This approach allows visualization of MTMR12 alongside its binding partners such as MTM1, without fluorophore spectrum overlap concerns.

• Multiplex Flow Cytometry: In flow cytometry applications, biotin-conjugated MTMR12 antibodies can be detected with streptavidin conjugates in channels that complement other directly-labeled antibodies. This enables simultaneous detection of MTMR12 expression alongside cell surface markers, activation indicators, or phosphorylation states .

• Proximity Ligation Assays (PLA): Biotin-conjugated MTMR12 antibodies can be paired with antibodies against suspected interaction partners in PLA workflows. This generates fluorescent signals only when proteins are within 40nm of each other, providing spatial resolution beyond conventional co-immunoprecipitation approaches.

• ChIP-seq Applications: For chromatin studies, biotin-conjugated antibodies facilitate more efficient chromatin pull-down, especially when combined with streptavidin magnetic beads, offering advantages similar to the 0.5-4.0 μg antibody per 1-3 mg protein ratio used in standard immunoprecipitation protocols .

What are the considerations for MTMR12 detection in disease model systems?

MTMR12 investigation in disease models requires specialized methodological considerations:

• Neuromuscular Disease Models: In models of centronuclear myopathy, MTMR12 detection should focus on skeletal muscle tissue using immunohistochemistry protocols optimized for muscle sections. The antibody concentration may need adjustment (typically 1:100-1:200) to penetrate the dense muscle tissue structure .

• Cancer Cell Lines: When investigating MTMR12 in cancer contexts, validation using multiple approaches is essential. The observed molecular weight may shift from the expected 86 kDa to approximately 73 kDa due to cancer-specific post-translational modifications or alternative splicing . Cell lines like K-562, HEK-293, and HeLa have been validated for MTMR12 detection and serve as appropriate positive controls .

• Developmental Models: For zebrafish models, which show 100% predicted reactivity with certain MTMR12 antibodies , specialized sample preparation is required. Whole-mount immunohistochemistry protocols should be modified with extended permeabilization steps (0.5% Triton X-100 for 1-2 hours) to ensure antibody penetration.

• Tissue-Specific Expression Analysis: MTMR12 expression varies by tissue type. When studying disease models, compare expression patterns against established baselines for the specific tissue, such as brain and lung tissues which have been validated for mouse MTMR12 detection .

The interplay between MTMR12 and other myotubularin family members in disease contexts should be considered when designing experimental approaches. Co-immunoprecipitation studies using biotin-conjugated MTMR12 antibodies can help elucidate altered protein interactions in disease states, following the recommended antibody-to-lysate ratios of 0.5-4.0 μg per 1-3 mg .