SLC2A2 Antibody, HRP conjugated

What is SLC2A2/GLUT2 and what are its primary physiological functions?

SLC2A2 (Solute carrier family 2, facilitated glucose transporter member 2), commonly known as GLUT2, is a facilitative glucose transporter protein with a molecular weight of approximately 60-70 kDa or 38-45 kDa depending on glycosylation state . This transmembrane protein serves dual roles in mammalian physiology - as both a transporter and a receptor/detector of glucose. As a transporter, GLUT2 mediates the bidirectional transfer of glucose across plasma membranes, particularly in hepatocytes . In pancreatic β cells, GLUT2 is responsible for glucose uptake and comprises part of the glucose-sensing mechanism . Additionally, GLUT2 participates alongside Na+/glucose cotransporters in the transcellular transport of glucose in the small intestine and kidney . This multifunctional protein plays a critical role in maintaining glucose homeostasis across multiple tissues and serves as an important target in metabolic disease research.

What are the optimal applications for SLC2A2 Antibody, HRP conjugated?

The SLC2A2 Antibody with HRP conjugation is optimized for several laboratory techniques with varying efficacy. For Western blot applications, the recommended dilution ranges from 1:100-1:1000, offering flexibility based on protein expression levels . For immunohistochemistry on paraffin-embedded sections (IHC-P), dilutions of 1:50-1:1000 are suggested, with the optimal concentration determined by target tissue type and fixation methods . The antibody performs effectively in ELISA applications as demonstrated by sandwich ELISA methods that utilize anti-SLC2A2 antibodies as capture antibodies . The HRP conjugation eliminates the need for secondary antibody incubation, offering workflow advantages in time-sensitive experiments. This direct detection system is especially beneficial for reducing background signal in tissues with high endogenous biotin or when working with samples that might cross-react with secondary antibodies.

What are the recommended storage conditions for maintaining SLC2A2 Antibody activity?

For optimal preservation of antibody function, SLC2A2 Antibody, HRP conjugated should be stored at -20°C or -80°C upon receipt . Research indicates that repeated freeze-thaw cycles significantly degrade antibody performance, particularly for conjugated antibodies where the activity of the HRP enzyme can be compromised . To minimize degradation, it is recommended to prepare small working aliquots before freezing . The antibody is typically supplied in a buffer containing preservatives like 0.03% Proclin 300, 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage . When handled properly, the antibody maintains validity for approximately 12 months . For short-term storage during experimental procedures, the antibody should be kept at 4°C and protected from light to prevent photobleaching of the HRP conjugate, which can affect detection sensitivity in subsequent applications.

Which species does the SLC2A2 Antibody reliably detect?

The commercially available SLC2A2 Antibody, HRP conjugated demonstrates confirmed reactivity across three mammalian species: human, mouse, and rat . This cross-reactivity is due to the high conservation of GLUT2 protein sequences across these species, making the antibody valuable for comparative studies. The antibody is typically raised against recombinant human Solute carrier family 2, facilitated glucose transporter member 2 protein (amino acids 67-174) , a region that maintains significant homology across rodent and human sequences. The conservation of epitopes in this region contributes to the antibody's consistent performance across species. When designing experiments utilizing this antibody for other species not explicitly listed in manufacturer specifications, researchers should consider performing preliminary validation tests, as sequence variations in specific regions might affect binding efficacy and signal intensity.

What is the molecular structure and topology of human GLUT2?

Human GLUT2, encoded by the SLC2A2 gene, is a 524-amino acid transmembrane protein belonging to the Major facilitator superfamily, specifically the Sugar transporter (TC 2.A.1.1) family, Glucose transporter subfamily . The topology of human GLUT2 has been predicted and refined through computational approaches using the Topo2 program based on sequences in UniProtKB/Swiss-Prot . The protein features multiple transmembrane domains forming a channel-like structure that facilitates glucose transport across the plasma membrane. Several glycosylation sites have been identified that may influence protein stability and function . The UniProt primary accession for human GLUT2 is P11168, with secondary accessions including A8K481, B2R936, B7Z547, F8W8V8, and Q9UCW9 . This detailed structural information is critical for understanding epitope accessibility when using antibodies for detection in different experimental contexts, particularly where protein conformation may be altered during sample preparation.

How do mutations in the SLC2A2 gene affect pancreatic β cell function and insulin secretion?

Research on SLC2A2 mutations has revealed critical insights into hGLUT2's dual functionality in pancreatic development and insulin regulation. Both gain and loss of function mutations in SLC2A2 have significant impacts on pancreatic β cell differentiation and insulin production pathways . Loss-of-function mutations are associated with Fanconi-Bickel Syndrome, characterized by impaired glucose homeostasis and abnormal glycogen accumulation . These mutations specifically disrupt the glucose-sensing mechanism in pancreatic β cells, compromising insulin secretion in response to hyperglycemia. Structure-function mapping of hGLUT2 has emphasized the protein's sugar receptor activity as distinct from its transport function, with specific amino acid regions differentially involved in these two roles . Experimental approaches using SLC2A2 antibodies have been instrumental in tracking the membrane expression profiles of wild-type versus mutant GLUT2 in hepatic and pancreatic β cells. Transport kinetics studies in Xenopus oocytes, combined with glucose-induced insulin secretion assays, have further delineated how specific mutations affect each function separately . These findings suggest that hGLUT2 represents a potential therapeutic target for stimulating pancreatic β cell differentiation and insulin secretion in diabetes treatment strategies.

What are the challenges in detecting GLUT2 in different tissue preparations?

Detecting GLUT2 across various tissue preparations presents several technical challenges that researchers should anticipate. First, the protein's membrane localization requires careful preservation of membrane integrity during sample preparation . Standard lysis buffers may inadequately solubilize membrane proteins, resulting in artificially low detection levels. Second, GLUT2 undergoes post-translational modifications, particularly glycosylation, which creates heterogeneity in molecular weight (observed at both 60-70 kDa and 38-45 kDa ranges) . This variability can complicate band interpretation in Western blots. Third, tissue-specific expression levels vary significantly - GLUT2 is abundant in liver hepatocytes but expressed at lower levels in pancreatic β cells, requiring different detection sensitivities . For formalin-fixed paraffin-embedded (FFPE) samples, antigen retrieval methods must be optimized to expose GLUT2 epitopes without destroying tissue morphology. In immunohistochemistry applications, the transmembrane nature of GLUT2 often necessitates membrane permeabilization procedures that must be balanced against maintaining structural integrity. Finally, endogenous biotin in liver and kidney tissues may cause high background when using avidin-biotin detection systems, making the direct HRP conjugation particularly valuable for these tissues.

How can I validate the specificity of SLC2A2 Antibody in my experimental model?

Rigorous validation of SLC2A2 antibody specificity is essential for generating reliable research data. A comprehensive validation approach should incorporate multiple complementary strategies. First, perform positive and negative control experiments using tissues or cell lines with known GLUT2 expression patterns - liver and pancreatic tissues as positive controls, and skeletal muscle (which primarily expresses GLUT4) as a negative control . Second, implement knockdown/knockout validation by comparing antibody signal in wild-type samples versus those with reduced or eliminated SLC2A2 expression through siRNA, shRNA, or CRISPR-Cas9 techniques . Third, conduct peptide competition assays where the antibody is pre-incubated with excess immunizing peptide (the SLC2A2 fragment used for antibody production, typically amino acids 67-174) - a specific antibody will show reduced or eliminated signal. Fourth, verify consistency across detection methods by confirming that your findings from Western blot correlate with immunostaining patterns or ELISA results using the same antibody . Finally, compare results with alternative antibodies targeting different GLUT2 epitopes; concordant results across different antibodies strongly support specificity. These combined approaches provide robust evidence for antibody specificity within your particular experimental context.

What are the optimal sample preparation techniques for detecting SLC2A2 in Western blots?

Successful detection of SLC2A2/GLUT2 in Western blots requires specialized sample preparation that preserves membrane protein integrity. Begin by extracting tissues or cells using membrane protein-optimized lysis buffers containing 1-2% of non-ionic detergents such as Triton X-100 or NP-40, which effectively solubilize membrane proteins while maintaining antibody-recognizable conformations . Include protease inhibitor cocktails to prevent degradation, and phosphatase inhibitors if phosphorylation status is relevant. When working with tissues rich in glycogen (such as liver), consider adding glycogen degrading enzymes to your lysis buffer to prevent smearing on gels. Importantly, avoid boiling samples before loading, as this can cause membrane protein aggregation; instead, heat samples to 37°C for 30 minutes or 50-60°C for 10 minutes . For electrophoresis, 8-10% SDS-PAGE gels optimally separate the 60-70 kDa glycosylated form of GLUT2 . Transfer to PVDF membranes (rather than nitrocellulose) often yields better results for hydrophobic membrane proteins. During blocking, avoid milk-based blockers which contain carbohydrates that may interfere with glucose transporter antibody binding; instead, use 3-5% BSA in TBS-T . For detection using the HRP-conjugated antibody, extended exposure times may be necessary, as membrane proteins often transfer less efficiently than soluble proteins.

What controls should be included when using SLC2A2 Antibody in knockout studies?

When designing knockout studies involving SLC2A2 Antibody, a comprehensive control strategy is essential for accurate data interpretation. Include wild-type (WT) samples processed identically to knockout (KO) samples as primary positive controls . Additionally, incorporate heterozygous samples when available, which should display intermediate signal intensity, confirming dose-dependent antibody response. For tissue-specific knockouts, include samples from non-targeted tissues that normally express GLUT2 (like liver in a pancreas-specific knockout) as internal positive controls . Loading controls should be carefully selected - traditional housekeeping proteins like β-actin or GAPDH are appropriate for assessing equal loading, but membrane protein controls such as Na+/K+ ATPase provide better normalization for membrane fraction enrichment variations. When using SLC2A2 Antibody, HRP conjugated in immunohistochemistry applications with knockout tissues, implement both primary antibody omission controls and isotype controls (rabbit IgG-HRP at equivalent concentrations) to assess non-specific binding . For functional validation, complement immunodetection with physiological assays measuring glucose transport activity or insulin secretion in response to glucose challenge . This multilayered control strategy enables confident attribution of observed phenotypes to GLUT2 absence rather than experimental artifacts or antibody cross-reactivity.

What is the recommended protocol for ELISA using SLC2A2 Antibody, HRP conjugated?

For optimal ELISA performance using SLC2A2 Antibody with HRP conjugation, a carefully standardized protocol should be followed. The sandwich ELISA method provides superior specificity and sensitivity for GLUT2 detection in complex biological samples . Begin with microplate preparation by coating wells with capture anti-SLC2A2 antibody (typically unconjugated) at 2-5 μg/ml in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C . Block remaining binding sites with 3% BSA in PBS for 1-2 hours at room temperature. Prepare a standard curve using recombinant human SLC2A2 protein in serial dilutions alongside properly diluted samples . Incubate standards and samples for 2 hours at room temperature with gentle shaking. After washing (PBS with 0.05% Tween-20, 4-5 cycles), add the HRP-conjugated SLC2A2 antibody at a 1:500-1:1000 dilution and incubate for 1-2 hours . Following another wash cycle, add TMB substrate solution and monitor color development - the blue product will turn yellow after adding the acidic stop solution . Measure optical density at 450nm using a microplate reader. For quantification, plot the standard curve and calculate SLC2A2 concentration in samples, noting that target protein concentration is proportional to OD450 values . This 4-hour assay provides reliable detection with a typical detection range of 0.1-8 ng/ml for SLC2A2 protein.

How can I troubleshoot non-specific binding when using SLC2A2 Antibody?

Non-specific binding is a common challenge when working with SLC2A2 antibodies that can be systematically addressed through methodical troubleshooting. If experiencing high background in Western blots, first optimize blocking conditions by testing alternative blocking agents - while 5% BSA is generally effective, for some tissue types, commercial blocking reagents may yield cleaner results . Increase the number and duration of washing steps, using TBS-T with 0.1% Tween-20 instead of the standard 0.05%. Titrate the primary antibody concentration - the recommended 1:100-1:1000 range for Western blots should be systematically tested to find the optimal signal-to-noise ratio for your specific samples . For immunohistochemistry applications showing excessive background, implement an avidin-biotin blocking step if using detection systems that might interact with endogenous biotin, particularly in liver, kidney, and pancreas tissues . Consider using Sudan Black B (0.1-0.3%) to reduce autofluorescence in fixed tissues. When multiple bands appear in Western blots, verify that additional bands aren't different glycosylation states of GLUT2 (60-70 kDa and 38-45 kDa) before assuming non-specificity. Additionally, pre-adsorbing the antibody with liver cell lysate from a species different from your experimental samples can reduce cross-reactivity with conserved epitopes that may be causing non-specific binding.

What is the best protocol for detecting SLC2A2 in FFPE tissue sections?

For optimal detection of SLC2A2 in formalin-fixed paraffin-embedded (FFPE) tissue sections, a specialized immunohistochemistry protocol is recommended. Begin with deparaffinization using standard xylene and graded ethanol series. Antigen retrieval is critical for SLC2A2 detection - heat-induced epitope retrieval in citrate buffer (pH 6.0) at 95-100°C for 20 minutes typically provides optimal results for membrane proteins . After cooling and washing in PBS, perform peroxidase blocking with 3% hydrogen peroxide for 10 minutes to minimize endogenous peroxidase activity. For tissues with high endogenous biotin (liver, kidney), include an avidin-biotin blocking step. Block non-specific binding sites with 5% normal goat serum in PBS containing 0.1% Triton X-100 for 1 hour at room temperature - the detergent facilitates antibody access to membrane-embedded GLUT2 . Apply the SLC2A2 Antibody, HRP conjugated at a 1:50-1:200 dilution (optimal dilution should be determined empirically) and incubate overnight at 4°C in a humidified chamber . After washing thoroughly (3-5 times, 5 minutes each) with PBS containing 0.05% Tween-20, proceed directly to chromogenic detection using DAB substrate, as secondary antibody incubation is unnecessary with HRP-conjugated primary antibodies . Counterstain with hematoxylin, dehydrate, clear, and mount. Expected GLUT2 localization includes plasma membranes of hepatocytes, pancreatic β cells, intestinal and renal epithelial cells, with particularly strong staining at basolateral membranes .

How can SLC2A2 Antibody be used to track changes in GLUT2 expression during diabetes progression?

The SLC2A2 Antibody, HRP conjugated provides a valuable tool for monitoring GLUT2 expression changes during diabetes progression through several methodological approaches. Longitudinal immunohistochemical studies of pancreatic tissues from diabetic animal models allow visualization of GLUT2 membrane localization changes in β cells at different disease stages . For quantitative assessment, Western blot analysis of membrane fractions extracted from pancreatic tissue or isolated islets provides measurable protein level changes over time . When designing such studies, standardize sample collection timing relative to feeding status, as GLUT2 expression and localization can be influenced by acute nutritional state . For enhanced sensitivity in detecting early changes, complement protein detection with mRNA analysis using RT-qPCR targeting SLC2A2 transcripts. In animal models, consider in vivo imaging using fluorescently-labeled SLC2A2 antibodies (converted from HRP-conjugated versions) to track real-time changes in the same subject over disease progression . Co-staining with insulin antibodies provides context for correlating GLUT2 alterations with β cell function . For human studies, analysis of GLUT2 in circulating extracellular vesicles derived from pancreatic β cells offers a less invasive biomarker approach. Importantly, establish clear quantification methods, ideally using automated image analysis software for immunohistochemistry or densitometry for Western blots, to ensure objective measurement of expression changes across disease time points.