slc16a12b Antibody

What is SLC16A12 and what cellular functions does it perform?

SLC16A12, also known as MCT12 (Monocarboxylate Transporter 12) or CRT2 (Creatine Transporter 2), belongs to the major facilitator superfamily and monocarboxylate porter family. It functions primarily as a transporter for creatine and its precursor guanidinoacetate (GAA). The transport activity is independent of resting membrane potential and extracellular Na⁺, Cl⁻, or pH levels. SLC16A12 plays a crucial role in the process of creatine biosynthesis and distribution throughout the body . This 53 kDa protein (observed at 53-56 kDa in experimental conditions) is a multi-pass membrane protein primarily localized to the basolateral cell membrane, with its cellular localization dependent on interaction with isoform 2 of BSG (Basigin) .

What tissues express SLC16A12 and where can positive controls be sourced?

Based on validated experimental evidence, SLC16A12 expression has been confirmed in several tissues that can serve as positive controls:

When designing experiments, these tissues can serve as appropriate positive controls for validating antibody performance and optimizing experimental conditions .

What are the recommended sample preparation methods for SLC16A12 detection?

For optimal detection of SLC16A12, sample preparation varies by technique. For immunohistochemistry, antigen retrieval is crucial and can be performed using either TE buffer (pH 9.0) or citrate buffer (pH 6.0) . The first method is generally preferred but both have been validated. For Western blotting, standard protein extraction protocols are suitable, with expected molecular weight between 53-56 kDa . When preparing samples for immunofluorescence, fixation with 4% paraformaldehyde followed by permeabilization with 0.1% Triton X-100 is recommended, though specific optimization may be necessary depending on tissue or cell type. For all applications, inclusion of protease inhibitors during sample preparation is advised to prevent protein degradation .

What are the optimal antibody dilutions for different experimental applications?

The recommended dilution ranges for SLC16A12 antibodies vary by application:

It is crucial to note that these ranges serve as starting points, and researchers should titrate the antibody in each testing system to obtain optimal results. The actual optimal dilution may be sample-dependent .

How should SLC16A12 antibodies be stored to maintain reactivity?

For maximum stability and performance, SLC16A12 antibodies should be stored at -20°C, where they remain stable for up to one year from the date of receipt . The antibodies are typically supplied in a liquid formulation containing PBS with 50% glycerol and 0.02% sodium azide (pH 7.3) , or in PBS with 50% glycerol, 0.5% BSA and 0.02% sodium azide . For the 20μl size antibodies from certain manufacturers, they may contain 0.1% BSA .

Importantly, repeated freeze-thaw cycles should be avoided as they can degrade antibody quality. For antibodies that will be used frequently, aliquoting is recommended, though some manufacturers note that aliquoting is unnecessary for -20°C storage .

What controls should be included when validating SLC16A12 antibody specificity?

When validating SLC16A12 antibody specificity, several controls should be implemented:

• Positive tissue controls: Use mouse kidney tissue for Western blot and human kidney, testis, or stomach cancer tissue for IHC based on validated results .

• Negative controls: Include tissues known not to express SLC16A12 or use secondary antibody-only controls to assess non-specific binding.

• Knockdown/knockout validation: Where possible, use SLC16A12 knockdown or knockout samples to confirm antibody specificity.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide before application to validate that binding is specifically blocked.

• Cross-reactivity assessment: Test the antibody against related proteins in the SLC16A family, particularly those with high sequence homology.

The antibody has been validated to detect endogenous levels of MCT12 protein, with demonstrated reactivity in human and mouse samples .

How can SLC16A12 antibodies be used to investigate creatine transport mechanisms?

To investigate SLC16A12-mediated creatine transport mechanisms, researchers can employ multiple complementary approaches:

• Co-localization studies: Use immunofluorescence with SLC16A12 antibodies in combination with markers for creatine or guanidinoacetate to visualize transport dynamics in live cells.

• Transport activity assays: Combine antibody-based detection of SLC16A12 with radiolabeled creatine uptake assays to correlate protein expression with functional transport activity.

• Mutation analysis: Utilize the antibody to detect expression levels of wild-type versus mutant SLC16A12 in conjunction with creatine transport assays to identify residues critical for transporter function.

• Interaction studies: Use co-immunoprecipitation with SLC16A12 antibodies to identify protein-protein interactions that might regulate creatine transport, especially focusing on the interaction with isoform 2 of BSG which is required for proper membrane localization .

• Subcellular fractionation: Apply Western blotting with the SLC16A12 antibody to different cellular fractions to track the transporter's trafficking pathway and membrane insertion dynamics.

These methods can provide insights into how SLC16A12 contributes to creatine biosynthesis and distribution, particularly considering that its transport function is independent of resting membrane potential and extracellular Na⁺, Cl⁻, or pH .

What considerations are important when using SLC16A12 antibodies in disease model research?

When applying SLC16A12 antibodies in disease model research, several critical considerations must be addressed:

• Genetic background effects: Different animal strains or human populations may show varied SLC16A12 expression patterns or splice variants that could affect antibody recognition. Validate antibody performance in each model system.

• Disease-specific modifications: Post-translational modifications of SLC16A12 may differ in disease states, potentially affecting antibody binding. Consider using multiple antibodies targeting different epitopes when studying pathological conditions.

• Expression changes: Quantification of SLC16A12 in disease models should account for potential changes in reference genes. Use multiple normalization controls for Western blotting or qPCR validation.

• Tissue-specific effects: Given SLC16A12's role in creatine transport and its association with juvenile cataracts, microcornea, and renal glucosuria , expression patterns may vary significantly between affected and unaffected tissues within the same organism.

• Cross-reactivity with therapeutic agents: If studying disease interventions, verify that treatments do not interfere with antibody binding or create artifacts.

Research involving SLC16A12 is particularly relevant to eye disorders and kidney function, as mutations in this gene have been associated with juvenile cataracts with microcornea and renal glucosuria .

How can phosphorylation status of SLC16A12 be assessed using available antibodies?

Assessment of SLC16A12 phosphorylation status requires a multifaceted approach, as current commercially available antibodies are not specifically designed to detect phosphorylated forms:

• Two-dimensional gel electrophoresis: Combine this with Western blotting using SLC16A12 antibodies to separate phosphorylated from non-phosphorylated forms based on isoelectric point shifts.

• Phosphatase treatment: Compare SLC16A12 migration patterns in Western blots of samples with and without phosphatase treatment to identify potential phosphorylated species.

• Phosphorylation-specific antibody development: Consider generating custom antibodies against predicted phosphorylation sites in SLC16A12 if commercially available options are insufficient.

• Mass spectrometry: Use immunoprecipitation with available SLC16A12 antibodies followed by mass spectrometry analysis to identify specific phosphorylation sites.

• Phosphorylation-dependent mobility shift assays: Utilize Phos-tag SDS-PAGE in combination with Western blotting using the SLC16A12 antibody to detect phosphorylated forms through mobility shifts.

When planning these experiments, researchers should consider that the SLC16A12 antibodies available (such as 20553-1-AP and STJ94047) recognize specific epitopes (STJ94047 targets amino acids 115-164 ), which may be affected by nearby phosphorylation events.

What are common issues when using SLC16A12 antibodies in IHC and how can they be resolved?

Common issues with SLC16A12 antibodies in immunohistochemistry and their solutions include:

For optimal IHC results with SLC16A12 antibodies, a critical step is proper antigen retrieval. The recommended approach is using TE buffer at pH 9.0, though citrate buffer at pH 6.0 can serve as an alternative . Titrate antibody dilutions starting with the manufacturer's recommended range and optimize blocking conditions to minimize background.

How can detection sensitivity be improved in Western blots using SLC16A12 antibodies?

To enhance detection sensitivity when working with SLC16A12 antibodies in Western blotting:

• Sample preparation optimization:

  • Enrich membrane fractions to concentrate SLC16A12 protein

  • Use mild detergents (like CHAPS or digitonin) that preserve membrane protein integrity

  • Include phosphatase inhibitors if studying phosphorylated forms

• Detection system selection:

  • Use high-sensitivity ECL substrates for chemiluminescence detection

  • Consider fluorescent secondary antibodies for improved quantification

  • Try signal amplification systems (e.g., biotin-streptavidin) for low abundance samples

• Blocking and antibody incubation:

  • Test different blocking agents (BSA vs. milk) to optimize signal-to-noise ratio

  • Extend primary antibody incubation to overnight at 4°C

  • Use antibody dilution within the recommended range (1:500-1:1000 or 1:500-1:2000 )

• Membrane treatment:

  • Consider PVDF over nitrocellulose for higher protein binding capacity

  • Optimize transfer conditions for high molecular weight proteins

  • Try wet transfer for more efficient transfer of membrane proteins

• Signal development:

  • Increase exposure time incrementally to capture weak signals

  • Use digital acquisition systems with adjustable sensitivity settings

The expected molecular weight for SLC16A12 is 53 kDa with observed bands at 53-56 kDa in experimental conditions , which can serve as a reference point for validating specific detection.

What strategies can address cross-reactivity with other SLC16A family members?

The SLC16A family contains multiple members with structural similarities, which can lead to antibody cross-reactivity. To address this issue:

• Epitope analysis: Review the immunogen sequence of the antibody (STJ94047 targets amino acids 115-164 ) and compare with other SLC16A family members to predict potential cross-reactivity.

• Validation in knockout/knockdown systems: Use cells or tissues with confirmed knockout/knockdown of SLC16A12 to verify that the signal disappears, confirming specificity.

• Peptide competition assays: Pre-incubate the antibody with the specific immunizing peptide to demonstrate signal reduction in a sequence-specific manner.

• Orthogonal detection methods: Complement antibody-based detection with RNA expression analysis (RT-PCR or RNA-Seq) to confirm correlation between protein and mRNA levels.

• Multiple antibody validation: Use antibodies targeting different epitopes of SLC16A12 to confirm consistent detection patterns.

• Immunoprecipitation-mass spectrometry: Perform IP with the SLC16A12 antibody followed by mass spectrometry to identify all proteins being captured, which can reveal off-target binding.

Both the Proteintech (20553-1-AP) and St John's Labs (STJ94047) antibodies have been affinity-purified using epitope-specific immunogens , which helps reduce but may not eliminate cross-reactivity with closely related family members.

How can SLC16A12 antibodies contribute to understanding disease mechanisms?

SLC16A12 antibodies provide valuable tools for investigating disease mechanisms, particularly in conditions associated with creatine metabolism and transport disorders:

• Cataract formation: Given the association between SLC16A12 mutations and juvenile cataracts with microcornea , antibodies can be used to track protein localization and expression in lens epithelial cells and compare patterns between normal and pathological samples.

• Renal function: SLC16A12's link to renal glucosuria suggests its role in kidney physiology. Antibodies can map expression patterns across different nephron segments and examine alterations in disease models.

• Creatine metabolism disorders: As a creatine transporter , SLC16A12 may be involved in broader creatine metabolism disorders. Antibodies can help establish tissue-specific expression patterns and functional correlations with creatine levels.

• Cancer research: Given its detection in stomach cancer tissue , investigating SLC16A12 expression changes in various cancer types could provide insights into metabolic reprogramming during carcinogenesis.

• Neurodegenerative conditions: Creatine's neuroprotective effects suggest potential roles for its transporters in brain health. SLC16A12 antibodies could help explore expression changes in neurodegenerative disease models.

Immunohistochemistry applications using these antibodies (dilution 1:50-1:500 or 1:100-1:300 ) could reveal tissue distribution changes in disease states, while Western blotting can quantify expression level differences between normal and pathological samples.

What are the challenges in developing isoform-specific antibodies for SLC16A12?

Developing truly isoform-specific antibodies for SLC16A12 presents several technical challenges:

• Sequence conservation: SLC16A family members share significant sequence homology, making it difficult to identify completely unique epitopes for antibody generation.

• Isoform identification: Complete characterization of all SLC16A12 splice variants is still evolving, complicating the design of isoform-specific antibodies.

• Post-translational modifications: Different cell types or conditions may produce SLC16A12 with varied post-translational modifications, affecting epitope availability and antibody recognition.

• Conformational epitopes: Membrane proteins like SLC16A12 have complex tertiary structures where conformational epitopes may be more specific than linear epitopes but are harder to target with antibodies.

• Validation complexity: Demonstrating true isoform specificity requires access to tissues or cells expressing only specific isoforms, which may not be readily available.

Current commercially available antibodies like 20553-1-AP and STJ94047 target specific regions of SLC16A12 (STJ94047 targets amino acids 115-164 ), but comprehensive isoform specificity testing data is limited. Researchers developing new antibodies should consider using recombinant expression systems with tagged isoforms for validation and employing epitope mapping to confirm specificity.