SLC5A1 Antibody, HRP conjugated

What is the recommended application range for SLC5A1 Antibody with HRP conjugation?

HRP-conjugated SLC5A1 antibodies are optimized for several applications with specific dilution recommendations:

• Western Blot: 1:300-5000 dilution

• ELISA: 1:500-1000 dilution

• IHC-P (Immunohistochemistry-Paraffin): 1:200-400 dilution

These ranges provide starting points for optimization. The specific antibody concentration varies by lot and manufacturer, so validation in your specific experimental system is recommended.

Which species reactivity has been confirmed for commercially available SLC5A1-HRP antibodies?

Most commercially available SLC5A1 antibodies with HRP conjugation have been validated for:

Cross-reactivity with other species may exist based on sequence homology but requires experimental validation .

What is the optimal storage condition for maintaining HRP-conjugated SLC5A1 antibody activity?

For maximum stability and preservation of activity:

• Store at -20°C for long-term storage

• Aliquot to avoid repeated freeze-thaw cycles

• For short-term storage (≤1 month after reconstitution), 2-8°C is acceptable

• Avoid exposure to light to preserve HRP activity

• Some formulations contain glycerol (40-50%) and preservatives like Proclin-300 (0.03%) or sodium azide (0.02-0.05%)

Proper storage is critical as HRP activity can diminish with improper handling.

How should I optimize antigen retrieval when using SLC5A1-HRP antibodies for IHC-P applications?

For IHC-P applications, optimal antigen retrieval significantly affects staining quality:

• Heat-Induced Epitope Retrieval (HIER) at pH 6.0 using citrate buffer is generally recommended

• Some antibodies perform better with Tris-EDTA buffer (pH 8.0)

• For human lung fibrosis tissue, Trilogy™ (EDTA-based, pH 8.0) buffer with 15 minutes retrieval has shown good results

• Temperature and duration optimization is critical: typically 95-100°C for 10-20 minutes

A comparison of different retrieval methods should be conducted during validation to determine optimal conditions for your specific tissue type.

What controls should be included when validating SLC5A1-HRP antibody specificity?

A comprehensive validation requires several controls:

• Positive tissue controls: Human duodenum shows high SLC5A1 expression and serves as an excellent positive control

• Negative tissue controls: Human cerebral cortex generally shows minimal SLC5A1 expression

• Absorption controls: Pre-incubation of antibody with immunizing peptide

• Secondary antibody controls: Omission of primary antibody

• Cross-validation: Compare results with another validated SLC5A1 antibody

• Knockout/knockdown validation: Using CRISPR or siRNA SLC5A1-depleted samples as described in pancreatic cancer studies

Correlating protein detection with RNA-seq data for SLC5A1 expression in the same tissues provides additional validation evidence .

How can SLC5A1-HRP antibodies be used to investigate SGLT1's role in pancreatic cancer progression?

Research has demonstrated SLC5A1's oncogenic role in pancreatic cancer, which can be investigated using SLC5A1 antibodies:

• Expression analysis: Compare SLC5A1 levels between pancreatic cancer tissue and adjacent non-cancerous tissue using IHC-P or Western blotting

• Functional studies: Assess glucose uptake in SLC5A1-knockdown pancreatic cancer cells using fluorescent glucose analogs (2-NBDG)

• Signaling pathway analysis: Investigate AMPK/mTOR pathway activation following SLC5A1 inhibition

• Protein-protein interactions: Examine SLC5A1 association with EGFR using co-immunoprecipitation followed by Western blot

Research findings show SLC5A1 knockdown reduces cellular glucose uptake, activates AMPK, and suppresses mTOR signaling, leading to reduced pancreatic cancer cell growth .

What protocol modifications are needed when investigating SLC5A1-EGFR protein interactions using co-immunoprecipitation?

When investigating SLC5A1-EGFR interactions, consider these methodological adaptations:

• Lysis buffer optimization: Use buffers containing 1% Triton X-100 or NP-40 that preserve membrane protein interactions

• Cross-linking (optional): Mild formaldehyde cross-linking (0.5-1%) may help stabilize transient interactions

• Antibody selection: Use non-conjugated SLC5A1 antibodies for immunoprecipitation

• Detection strategy:

  • Immunoprecipitate with anti-SLC5A1 and detect EGFR by Western blot

  • Reverse approach: immunoprecipitate with anti-EGFR and detect SLC5A1

• Controls: Include IgG control immunoprecipitation and input sample controls

• Validation: Confirm interactions by reciprocal co-immunoprecipitation or proximity ligation assay

Research has established a correlation between SLC5A1 and EGFR expression in pancreatic cancer patients (P=0.0035), with direct protein interaction confirmed by co-immunoprecipitation .

What are the most common causes of non-specific background when using SLC5A1-HRP antibodies in Western blotting?

Non-specific background can compromise data interpretation. Common causes and solutions include:

The observed molecular weight of SLC5A1 should be approximately 73 kDa, though post-translational modifications may alter the apparent molecular weight .

How should I interpret discrepancies between expected and observed SLC5A1 expression patterns across different tissues?

When encountering unexpected expression patterns:

• Compare with literature data: SLC5A1 is highly expressed in intestinal epithelium (duodenum) but lower in cerebral cortex

• Verify antibody specificity: Use multiple antibodies targeting different epitopes (e.g., NBP2-38748 and NBP2-33629 for orthogonal validation)

• Consider post-translational modifications: These can affect antibody recognition

• Evaluate RNA expression: Compare protein expression with RNA-seq data for the same tissues

• Assess experimental conditions: Sub-optimal conditions may lead to false negative results

• Consider biological variables: Expression can vary with disease state, physiological conditions, or genetic background

How can SLC5A1-HRP antibodies be utilized in investigating genetic variants associated with congenital glucose-galactose malabsorption?

SLC5A1 mutations cause congenital glucose-galactose malabsorption, a rare autosomal recessive disorder. Antibody-based approaches can help investigate these variants:

• Expression analysis: Compare expression levels of wild-type vs. mutant SLC5A1 in patient samples

• Localization studies: Examine subcellular localization changes in mutant proteins using IHC or ICC

• Functional validation: After site-directed mutagenesis introduction of specific variants, detect protein expression levels by Western blot

• Trafficking studies: Determine if mutations affect membrane localization using cell-surface biotinylation followed by antibody detection

• Structure-function correlations: Map epitope recognition to help understand structural impacts of mutations

Research on Turkish patients has identified several SLC5A1 mutations through molecular studies that can be further characterized using antibody-based methods .

What methodological considerations are important when using SLC5A1-HRP antibodies to study the relationship between glucose transport and cancer metabolism?

When investigating SLC5A1's role in cancer metabolism:

• Physiological glucose conditions: Compare antibody-detected SLC5A1 expression under varying glucose concentrations (0.5mM, 5mM, 25mM, 50mM)

• Metabolic stress conditions: Assess SLC5A1 expression under hypoxia, nutrient deprivation, or pharmacological AMPK activation

• Multi-parameter analysis: Combine SLC5A1 detection with markers of cellular metabolism (HIF-1α, GLUT1) or signaling pathways (p-AMPK, p-mTOR)

• In vivo tumor models: Use SLC5A1 antibodies for IHC analysis of xenograft tumors before and after glucose transport inhibition

• Patient sample stratification: Correlate SLC5A1 expression with clinical outcomes and metabolic parameters

Research has demonstrated that SLC5A1 knockdown reduces glucose uptake in pancreatic cancer cells, triggers AMPK activation, and suppresses mTOR signaling, ultimately inhibiting cancer cell growth .

What validation standards should be applied when using different SLC5A1-HRP antibody lots for longitudinal studies?

To ensure data consistency across different antibody lots:

• Lot-to-lot validation: Compare new lots against previous lots using:

  • Western blot of reference samples

  • Comparative IHC/ICC on standard positive controls

  • Titration curves to ensure similar sensitivity

• Reference standard inclusion: Include a consistent positive control sample in each experiment

• Quantitative calibration: Use standard curves with known quantities of recombinant SLC5A1

• Documentation: Maintain detailed records of lot numbers, dilutions, and experimental conditions

• Signal normalization: Normalize signal intensity to housekeeping proteins or total protein staining

Manufacturers typically perform lot-to-lot testing, but researcher validation is essential for critical applications.

How do I accurately document the specificity and sensitivity parameters of SLC5A1-HRP antibodies in my research publications?

For comprehensive antibody reporting in publications:

• Complete antibody information:

  • Manufacturer and catalog number

  • Clone/lot number

  • Host species and antibody type

  • Immunogen details (if available)

• Validation evidence:

  • Positive and negative controls used

  • Knockout/knockdown validation

  • Cross-reactivity testing

• Experimental conditions:

  • Exact dilutions used

  • Incubation times and temperatures

  • Blocking reagents

  • Antigen retrieval methods

• Detection systems:

  • Enhanced chemiluminescence (ECL) details

  • Image acquisition parameters

• Quantification methods:

  • Software used

  • Normalization approach

Following these documentation standards improves reproducibility and facilitates accurate interpretation by the research community.