SLC16A12 Antibody, HRP conjugated

What is SLC16A12 and what cellular functions does it perform?

SLC16A12, also known as Monocarboxylate transporter 12 (MCT12), functions as a proton-linked monocarboxylate transporter that mediates creatine transport across the plasma membrane . It belongs to the major facilitator superfamily and Monocarboxylate porter family (TC 2.A.1.13) . This protein catalyzes the rapid transport of various monocarboxylates across cellular membranes, including metabolites such as lactate and pyruvate, which play critical roles in cellular metabolism . SLC16A12's importance extends to multiple research areas including neuroscience, cancer metabolism, and signal transduction pathways .

What is the molecular weight and structure of SLC16A12 protein?

SLC16A12 has a calculated molecular weight of 53 kDa, though in experimental settings it is typically observed between 53-56 kDa . Structurally, the full-length protein contains multiple transmembrane domains that are essential for its transport function. The protein's tertiary structure resembles other monocarboxylate transporters, with the Q215 residue located in the large intracellular loop between the sixth and seventh transmembrane domains . The C-terminal cytoplasmic tail (amino acids 478-500) has been used as an epitope for antibody generation, suggesting its accessibility in the folded protein .

In which tissues is SLC16A12 expression most prominent?

SLC16A12 expression has been documented in several tissues, with particularly strong detection in:

This tissue distribution pattern suggests SLC16A12's physiological relevance in specialized transport processes across these diverse organ systems .

How does SLC16A12 trafficking occur in normal versus mutated states?

Studies comparing wildtype and mutated SLC16A12 have revealed critical insights into protein trafficking mechanisms. When exogenously expressed, full-length MCT12 (wildtype) is properly trafficked to the plasma membrane, whereas the truncated MCT12:214Δ protein (containing a mutation) is retained in the endoplasmic reticulum (ER) . In co-expression experiments mimicking the heterozygous patient genotype, the truncated protein remains trapped in the ER while the full-length MCT12 successfully reaches the plasma membrane .

Furthermore, research has identified that MCT12 requires the chaperone protein CD147 for proper trafficking to the cell surface, similar to other MCT isoforms . This dependency on chaperone proteins represents an additional regulatory mechanism controlling SLC16A12 localization and function.

What are the implications of SLC16A12 mutations in disease pathology?

SLC16A12 mutations have been associated with juvenile cataract formation. The c.643C>T mutation creates a premature stop codon (p.Q215X) resulting in a truncated protein with only the first six transmembrane domains . This mutation appears to cause disease through protein misfolding rather than haploinsufficiency, as complete loss of MCT12 in rat models (Slc16a12^-/-) did not result in detectable ocular phenotypes .

The dominant inheritance pattern observed in patients with this mutation suggests that the retained, misfolded protein may interfere with normal cellular processes, potentially through:

• ER stress responses triggered by accumulated misfolded proteins

• Disruption of normal metabolite transport

• Interference with other membrane transport systems

These findings position SLC16A12 as an important research target for understanding metabolic transport disorders and their connection to tissue-specific pathologies .

What are the optimal conditions for using SLC16A12 Antibody, HRP conjugated in different experimental applications?

For all applications, it's recommended to perform initial titration experiments to determine optimal antibody concentration for specific experimental conditions . When using the HRP-conjugated version, no secondary antibody is required, which can reduce background and cross-reactivity in sensitive applications .

What are the recommended storage and handling procedures to maintain antibody activity?

To maintain optimal activity of SLC16A12 Antibody, HRP conjugated:

• Upon receipt, store at -20°C or -80°C

• Avoid repeated freeze-thaw cycles, which can degrade antibody activity

• For longer-term storage, consider aliquoting the antibody into single-use volumes

• When using lyophilized formats, reconstitute in the recommended buffer (e.g., 50 μL distilled water for a final concentration of 1 mg/mL)

• Store in buffers containing stabilizers such as glycerol (50%) or sucrose (2%)

• Some formulations contain preservatives like 0.03% Proclin 300 or 0.02% sodium azide to prevent microbial contamination

Proper storage and handling are critical for maintaining antibody specificity and sensitivity, particularly for conjugated antibodies where both protein integrity and enzyme activity must be preserved .

How can researchers validate the specificity of SLC16A12 antibodies in their experimental system?

Validating antibody specificity is crucial for reliable results. For SLC16A12 antibodies, consider these approaches:

• Positive and negative controls:

  • Use tissues with known expression (kidney, eye tissues) as positive controls

  • Include knockout/knockdown samples as negative controls where possible

  • The Slc16a12^-/- rat model could serve as an excellent negative control

• Verification with overexpression systems:

  • Transiently transfect cells with MCT12-GFP and confirm co-localization with the antibody

  • This approach confirmed specificity of anti-peptide antibodies against amino acids 478-500 of human MCT12

• Peptide competition assays:

  • Pre-incubate the antibody with immunizing peptide to block specific binding

  • Example: Antibodies generated against the synthetic oligopeptide SDPKLQLWTNGSVAYSVARELDQ (amino acids 478-500) should show reduced signal when pre-blocked

• Cross-validation with multiple antibodies:

  • Compare results using antibodies targeting different epitopes of SLC16A12

  • Compare HRP-conjugated versus unconjugated primary antibodies for consistent patterns

What experimental considerations should researchers address when studying SLC16A12 trafficking in cellular models?

When investigating SLC16A12 trafficking mechanisms:

• Selection of appropriate cell models:

  • Consider cell lines that naturally express SLC16A12 versus transfected systems

  • HEK-293 cells have been successfully used for exogenous expression studies

• Co-expression with trafficking partners:

  • Include studies of CD147, which is required for MCT12 trafficking to the cell surface

  • Consider co-immunoprecipitation experiments to identify additional interaction partners

• Subcellular localization techniques:

  • Use organelle-specific markers to distinguish plasma membrane localization from ER retention

  • For mutant proteins, validate ER retention with ER markers and stress response indicators

• Trafficking dynamics:

  • Implement pulse-chase experiments to track protein movement through cellular compartments

  • Consider temperature-controlled experiments (e.g., 20°C blocks) to arrest trafficking at specific steps

• Mutation effects:

  • When studying mutations like c.643C>T, design constructs that allow visualization of protein trafficking

  • Use fluorescent protein tags (like GFP) positioned to avoid interference with trafficking signals

What strategies can resolve poor signal or high background when using SLC16A12 Antibody, HRP conjugated?

When troubleshooting, it's advisable to run appropriate positive controls such as mouse kidney tissue for Western blot or human kidney tissue for IHC to validate that your experimental conditions support SLC16A12 detection .

How can researchers optimize detection of SLC16A12 in different sample types?

For tissue samples:

• For IHC applications, perform antigen retrieval with TE buffer pH 9.0 as the primary method, or alternatively try citrate buffer pH 6.0

• Different fixation protocols may affect epitope accessibility; compare paraformaldehyde versus formalin fixation

• Consider tissue-specific optimization: kidney tissues generally show good detection, while other tissues may require modified protocols

For cell culture samples:

• When studying overexpression systems, timing of sample collection after transfection is critical (typically 24-48 hours)

• For endogenous detection, select cell lines known to express SLC16A12

• Consider membrane fractionation to enrich for plasma membrane proteins

For protein extraction:

• Use detergent combinations optimized for membrane proteins (e.g., RIPA buffer with additional non-ionic detergents)

• Avoid excessive heat during sample preparation which may cause aggregation of membrane proteins

• Include protease inhibitors to prevent degradation of the target protein

By systematically addressing these experimental variables, researchers can optimize detection protocols for their specific experimental systems and research questions related to SLC16A12 .