MCTP2 Antibody

What is MCTP2 and what cellular functions does it perform?

MCTP2 is a transmembrane protein containing multiple C2 domains involved in intercellular signal transduction and synapse function. The full-length MCTP2 gene spans approximately 180 kilobases of genomic DNA and contains 22 coding exons, encoding a protein with 878 amino acid residues with a molecular weight of 100 kDa. The protein has a distinctive structure with three C2 domains and two transmembrane regions (TMRs) . MCTP2 is localized in discrete regions of the endoplasmic reticulum (ER) membrane and plays a significant role in lipid droplet (LD) biogenesis, promoting LD formation at specialized ER subdomains . Recent research has also implicated MCTP2 mutations in congenital prosopagnosia (face blindness), suggesting its importance in neural development and function, particularly in the right fusiform face area (rFFA) of the brain .

What applications are validated for MCTP2 antibody?

The MCTP2 antibody (17578-1-AP) has been validated for multiple research applications including Western Blot (WB), Immunohistochemistry (IHC), and ELISA . Western blotting detects MCTP2 protein at approximately 100 kDa, matching its calculated molecular weight. For immunohistochemistry, the antibody has been successfully used on paraffin-embedded human brain tissue, demonstrating specific localization patterns . The antibody shows reactivity with human, mouse, and rat samples, making it versatile for comparative studies across these species . When conducting these applications, it is important to follow the recommended protocols and optimization procedures to ensure specific and reproducible results.

What are the recommended storage conditions and handling practices for MCTP2 antibody?

MCTP2 antibody should be stored at -20°C, where it remains stable for one year after shipment. The storage buffer typically consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For the 17578-1-AP antibody, aliquoting is unnecessary for -20°C storage, which simplifies handling procedures . Some preparations (20μl sizes) contain 0.1% BSA as a stabilizer . The SAB MCTP2 antibody is also recommended to be stored at -20°C or below, with instructions to avoid cycles of freezing and thawing to maintain antibody integrity . For extended storage, it's recommended to aliquot contents and freeze, while dilution should only be performed immediately prior to use . These storage conditions ensure optimal antibody performance and prevent degradation that could compromise experimental results.

What are the optimal dilution ratios for different applications of MCTP2 antibody?

For Western Blot applications, the recommended dilution range for MCTP2 antibody (17578-1-AP) is 1:500-1:2000 . This range allows researchers to optimize signal-to-noise ratio based on their specific experimental conditions and sample types. For Immunohistochemistry, a more concentrated application is typically required, with recommended dilutions ranging from 1:20-1:200 . The appropriate dilution may vary depending on tissue type, fixation method, and detection system. For instance, in validation studies, immunohistochemical analysis of paraffin-embedded human brain was successfully performed using a 1:100 dilution . It is strongly recommended to titrate the antibody in each testing system to obtain optimal results, as the performance can be sample-dependent . Reference to the validation data gallery provided by manufacturers can provide insight into expected results at various dilutions.

How should antigen retrieval be optimized for MCTP2 immunohistochemistry?

For MCTP2 immunohistochemistry, antigen retrieval optimization is critical for successful staining. The primary recommendation is to use TE buffer at pH 9.0 for antigen retrieval . This alkaline pH helps to break protein cross-links formed during formalin fixation, thereby enhancing epitope accessibility. Alternatively, citrate buffer at pH 6.0 can be used if TE buffer does not yield satisfactory results . The choice between these methods may depend on the specific tissue type and fixation conditions. For human brain tissue, which has been validated as a positive control, the antigen retrieval method should be carefully selected and optimized. Researchers should consider performing parallel experiments with both retrieval methods to determine which provides superior staining with minimal background. Temperature, duration of heating, and cooling periods are additional variables that may require optimization for specific tissue samples.

What positive controls should be used for validating MCTP2 antibody experiments?

For Western Blot applications, mouse liver tissue and human placenta tissue have been validated as reliable positive controls for MCTP2 antibody . These tissues show consistent expression of MCTP2 at the expected molecular weight of approximately 100 kDa. For immunohistochemistry applications, human brain tissue serves as an appropriate positive control . This aligns with findings that MCTP2 is expressed in the right fusiform gyrus of the brain, which contains the right Fusiform Face Area (rFFA) . When establishing experimental protocols, researchers should first validate their antibody using these recommended positive controls before proceeding to experimental samples. This validation step ensures that any negative results in experimental samples are due to biological differences rather than technical failures. The inclusion of these positive controls in each experimental run also provides an internal quality control measure for assessing staining consistency across experiments.

How does MCTP2 contribute to lipid droplet biogenesis and membrane dynamics?

MCTP2, along with its related protein MCTP1, localizes to discrete regions of the endoplasmic reticulum (ER) membrane where it promotes the formation of lipid droplets (LDs) . High-resolution live-cell microscopy has demonstrated that nascent LDs form specifically at MCTP subdomains within the ER . The function of MCTP2 in LD biogenesis appears to be regulated by its structural domains: the reticulon homology domains (RHDs) promote LD formation, while the C2 domains regulate LD size, likely by mediating ER-LD contacts . The transmembrane domains (TMDs) of MCTPs share structural similarities with reticulon homology domains (RHDs) found in several reticulon and reticulon-like proteins, containing two hydrophobic regions 30-37 amino acids in length . This suggests that MCTP2 may play a role in shaping ER membrane curvature, which is essential for LD budding. Research examining MCTP2's involvement in membrane dynamics requires sophisticated imaging techniques such as high-resolution confocal microscopy or electron microscopy to visualize these subcellular structures and dynamic processes.

What is the significance of MCTP2 mutations in congenital prosopagnosia and cardiac malformations?

Genetic research has revealed that mutations in MCTP2 are associated with congenital prosopagnosia (CP), a condition characterized by the inability to recognize faces . Whole-exome sequencing (WES) identified a specific mutation (c.T2147G; p.I716S) in MCTP2 that co-segregated with CP in affected family members . The isoleucine at position 716, located in the first transmembrane region of MCTP2, is highly conserved across primate species, suggesting functional importance . Expression data from the Allen Human Brain Atlas supports MCTP2 expression in the right fusiform gyrus, which contains the right Fusiform Face Area (rFFA), a core brain region consistently activated by face recognition tasks . Additionally, MCTP2 has been implicated as a potential genetic cause of coarctation of the aorta (CoA) and related cardiac malformations . These findings suggest that MCTP2 plays crucial roles in both neurological development and cardiac morphogenesis, possibly through its involvement in intercellular signaling and membrane dynamics. Researchers investigating these conditions should consider using the MCTP2 antibody in combination with genetic analysis to correlate protein expression with specific mutations.

How can researchers differentiate between MCTP2 and other C2 domain-containing proteins?

Differentiating MCTP2 from other C2 domain-containing proteins requires a multifaceted approach incorporating both structural analysis and functional characterization. MCTP2 has a distinctive structure with three C2 domains (C2A, C2B, and C2C) at the N-terminus and transmembrane domains at the C-terminus . Unlike many C2 domain proteins, MCTPs contain a unique combination of multiple C2 domains and transmembrane regions. When using antibodies, researchers should verify the specificity by confirming that the immunogen used to generate the MCTP2 antibody is unique to this protein. For instance, the 17578-1-AP antibody was raised against a specific MCTP2 fusion protein (Ag11089) . Computational approaches can also aid in differentiating MCTP2 from related proteins; tools like HHpred have been used to analyze the structural similarities between MCTP TMDs and reticulon homology domains . Functionally, while many C2 domain proteins are involved in calcium-dependent membrane binding, the specific localization of MCTP2 to discrete ER regions and its role in lipid droplet formation provide distinguishing characteristics . For definitive differentiation, researchers might employ techniques such as immunoprecipitation followed by mass spectrometry, or use specific genetic knockdown approaches to confirm antibody specificity.

Why might Western blot results using MCTP2 antibody show inconsistent band patterns?

Inconsistent band patterns in Western blots using MCTP2 antibody can stem from multiple factors requiring systematic troubleshooting. Given that MCTP2 has a calculated molecular weight of 100 kDa and this matches the observed molecular weight in validated samples , significant deviations may indicate technical issues or biological variations. Protein degradation can occur if samples are not properly handled or if protease inhibitors are insufficient, resulting in lower molecular weight bands. Conversely, post-translational modifications like glycosylation, phosphorylation, or ubiquitination can cause shifts to higher molecular weights. Alternative splicing of MCTP2, which has 22 coding exons , might produce isoforms of different sizes. The antibody concentration is also critical; the recommended dilution of 1:500-1:2000 for Western blotting should be optimized for each experimental system . Sample preparation variables, including buffer composition, denaturation conditions, and reducing agent concentration, can affect protein conformation and antibody accessibility. To troubleshoot inconsistent results, researchers should compare their samples with validated positive controls such as mouse liver tissue or human placenta tissue , and systematically vary experimental conditions while maintaining consistent sample handling.

How can high background issues be resolved when using MCTP2 antibody in immunohistochemistry?

High background in immunohistochemistry using MCTP2 antibody can compromise data interpretation and requires methodical resolution. First, optimize the antibody dilution, testing various concentrations within the recommended range of 1:20-1:200 . Antigen retrieval methods significantly impact background; compare the suggested TE buffer (pH 9.0) against the alternative citrate buffer (pH 6.0) to determine which produces cleaner results with your specific tissue. Blocking procedures should be thorough, using appropriate blocking agents compatible with your detection system and extending blocking times if necessary. Washing steps are critical; insufficient washing after primary and secondary antibody incubations often contributes to high background. For MCTP2 detection in tissues with intrinsic autofluorescence or high endogenous peroxidase activity, consider additional quenching steps before antibody application. Secondary antibody cross-reactivity can also generate background; use species-specific secondary antibodies and consider pre-absorbing them against tissue proteins. If background persists despite these measures, alternative detection systems or visualization methods may be necessary. Finally, include appropriate negative controls (primary antibody omission, isotype controls) alongside your experimental samples to accurately distinguish between specific signal and background.

What factors might affect the specificity of MCTP2 antibody in experimental applications?

The specificity of MCTP2 antibody can be influenced by several factors that researchers should carefully control. The antibody's polyclonal nature (17578-1-AP is rabbit polyclonal IgG) means it contains antibodies recognizing multiple epitopes on MCTP2, which increases sensitivity but may reduce specificity compared to monoclonal antibodies. Cross-reactivity with structurally similar proteins, particularly other C2 domain-containing proteins or related MCTP family members like MCTP1, can occur. This risk increases in experimental conditions that denature proteins or alter their conformation. Fixation methods significantly impact epitope preservation; overfixation can mask epitopes while underfixation may not adequately preserve protein structure. The antibody concentration directly affects specificity; higher concentrations increase sensitivity but may reduce specificity. For optimal results, titrate within the recommended ranges (1:500-1:2000 for WB, 1:20-1:200 for IHC) . Sample quality and preparation also influence specificity; degraded samples or improper tissue processing can lead to non-specific binding. To confirm antibody specificity, consider additional validation techniques such as using MCTP2 knockout/knockdown controls, peptide competition assays, or comparing results with a second MCTP2 antibody targeting a different epitope. Always include positive controls (mouse liver tissue, human placenta tissue for WB; human brain tissue for IHC) to verify proper antibody function.

How might MCTP2 antibody applications extend to studying neurodevelopmental disorders?

The discovery of MCTP2 mutations in congenital prosopagnosia (face blindness) opens significant research avenues for studying neurodevelopmental disorders using MCTP2 antibody. Since MCTP2 is expressed in the right fusiform gyrus containing the right Fusiform Face Area (rFFA) , researchers can employ immunohistochemistry with MCTP2 antibody to examine protein expression patterns in neurodevelopmental conditions affecting face processing. Brain tissue immunostaining at 1:20-1:200 dilutions can reveal alterations in MCTP2 localization or expression levels in affected individuals. Western blot analysis of brain tissue samples can quantify potential expression differences between control and disorder-affected tissues. The antibody's validated reactivity across human, mouse, and rat samples enables translational research using animal models of neurodevelopmental disorders. Researchers might combine MCTP2 immunostaining with markers for synaptic proteins to investigate how MCTP2 mutations affect synapse formation and function, given MCTP2's role in intercellular signal transduction and synapse function . Future studies could correlate MCTP2 expression patterns with specific genetic variants identified in patient populations, potentially revealing structure-function relationships that explain clinical phenotypes. This approach may extend beyond prosopagnosia to other face processing disorders or broader neurodevelopmental conditions affecting brain regions where MCTP2 is functionally significant.