SCNN1G Antibody

What is SCNN1G and why is it an important research target?

SCNN1G (sodium channel epithelial 1 subunit gamma) encodes the gamma subunit of the epithelial Na+ channel (ENaC). This nonvoltage-gated, amiloride-sensitive sodium channel is crucial for controlling fluid and electrolyte transport across epithelia in numerous organs. The ENaC channel comprises three subunits (alpha, beta, and gamma) that form heteromeric complexes, with SCNN1G providing the gamma component .

ENaC channels are primarily expressed in the apical membrane of salt-absorbing epithelia of the kidney, distal colon, and lung. They play essential roles in:

• Maintaining salt and fluid homeostasis across epithelial tissues

• Regulating blood pressure through sodium reabsorption

• Controlling airway surface liquid homeostasis for proper mucus clearance

• Mediating aldosterone-dependent sodium reabsorption in the distal nephron

Mutations in SCNN1G have been associated with conditions like Liddle syndrome and pseudohypoaldosteronism type 1, making it an important target for both basic and translational research .

What applications are SCNN1G antibodies typically used for in research?

Based on available commercial antibodies and research literature, SCNN1G antibodies are primarily utilized in:

For IHC applications, antigen retrieval is typically recommended with TE buffer (pH 9.0) or citrate buffer (pH 6.0). Positive control tissues include human esophagus cancer specimens and mouse cerebellum tissue .

What are the basic storage and handling considerations for SCNN1G antibodies?

For optimal performance and longevity of SCNN1G antibodies:

• Store at -20°C in aliquots to avoid repeated freeze-thaw cycles

• Most commercial SCNN1G antibodies remain stable for one year when properly stored

• Typical storage buffer consists of PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Some preparations may contain 0.1% BSA for additional stability

• Ship on blue ice or equivalent cold chain

• Allow antibody to equilibrate to room temperature before opening vial

• Brief centrifugation may be needed to collect solution at the bottom of the vial

Proper handling ensures antibody stability and optimal performance in downstream applications .

How should researchers select the most appropriate SCNN1G antibody for their specific research application?

Selection criteria should be based on:

• Epitope consideration: Different antibodies recognize different regions of SCNN1G. For example:

  • Some antibodies target fusion proteins of human SCNN1G (aa630-649)

  • Others target specific domains like the extracellular or transmembrane regions

  • Consider whether your experiment requires detection of specific domains or full-length protein

• Species reactivity: Different antibodies show varying cross-reactivity:

  • Some react with human, mouse, and rat SCNN1G

  • Others may only react with human and pig SCNN1G

  • Confirm species homology if working with non-human models

• Validated applications: Choose antibodies specifically validated for your intended application:

  • Some antibodies work well for IHC but fail in WB applications

  • Antibody PA1-922, for example, works for immunoprecipitation but fails in Western blot procedures

• Clonality considerations:

  • Polyclonal antibodies offer broader epitope recognition but potential batch-to-batch variability

  • All commercial SCNN1G antibodies in the search results are polyclonal, raised in rabbits

• Molecular weight detection: SCNN1G has a calculated molecular weight of 74 kDa, but observed weights can range from 70-85 kDa due to post-translational modifications .

Always perform validation studies with proper positive and negative controls in your specific experimental system.

What controls should be included when using SCNN1G antibodies in research applications?

Rigorous experimental design should include the following controls:

For Western Blotting:

• Positive control tissues/cells: A431 cells, COLO 320 cells, PC-3 cells, or pig kidney tissue

• Negative control: Tissues or cell lines known not to express SCNN1G

• Loading control: Antibody against housekeeping proteins (β-actin, GAPDH)

• Peptide competition: Pre-incubation of antibody with immunizing peptide should abolish specific signal

For Immunohistochemistry:

• Positive control tissues: Mouse cerebellum tissue, human esophagus cancer

• Isotype control: Normal rabbit IgG at the same concentration

• Antigen retrieval controls: Compare TE buffer (pH 9.0) and citrate buffer (pH 6.0)

• Antibody concentration gradient: Test dilution range (e.g., 1:500, 1:1000, 1:2000)

• Peptide competition control: As seen in the Feng et al. research, where including the antigenic peptide significantly reduced immunostaining intensity

For Immunoprecipitation:

• Input control: Original lysate before immunoprecipitation

• Negative control IP: Non-specific IgG from same species as primary antibody

• Reverse IP: IP with antibody against interacting protein partner, followed by SCNN1G detection

These controls ensure specificity and reliability of results while helping identify potential false positives or negatives.

What are the optimal sample preparation methods for detecting SCNN1G in different tissue types?

Sample preparation varies by tissue type and intended application:

For epithelial tissues (kidney, lung, colon):

• Fresh tissues should be rapidly fixed in 4% paraformaldehyde or 10% neutral buffered formalin

• For membrane protein preservation, avoid excessive fixation (12-24 hours optimal)

• Cryosections may better preserve antigenicity but have poorer morphology

• Paraffin embedding requires careful antigen retrieval (see protocol below)

Antigen retrieval protocol for FFPE tissues:

• Deparaffinize and rehydrate sections through xylene and graded alcohols

• Heat-induced epitope retrieval with TE buffer (pH 9.0) in pressure cooker for 20 minutes

• Alternative: use citrate buffer (pH 6.0) if TE buffer yields high background

• Allow slides to cool to room temperature (approximately 20 minutes)

• Block endogenous peroxidase activity with 3% H₂O₂ for 10 minutes

• Block non-specific binding with 5-10% normal serum in PBS for 1 hour

For cell lines:

• Culture cells to 70-80% confluence

• Wash with ice-cold PBS

• For WB: Lyse in RIPA buffer containing protease inhibitors

• For IP: Use milder lysis buffer (e.g., NP-40-based) to preserve protein-protein interactions

• For IF: Fix with 4% paraformaldehyde for 15 minutes at room temperature

Membrane protein extraction protocols may be particularly important for SCNN1G detection due to its localization in the cell membrane .

How can researchers effectively detect heteromeric ENaC complexes that include the SCNN1G subunit?

Detecting intact heteromeric ENaC complexes requires specialized techniques:

Co-immunoprecipitation approach:

• Use mild lysis buffers (1% digitonin or 0.5% NP-40) to preserve protein-protein interactions

• Immunoprecipitate with antibodies against one subunit (e.g., anti-SCNN1G)

• Probe Western blots with antibodies against other subunits (SCNN1A, SCNN1B)

• Include controls for non-specific binding

Blue native PAGE:

• Solubilize membranes with mild detergents (digitonin or n-dodecyl-β-D-maltoside)

• Separate native complexes by blue native PAGE

• Perform second dimension SDS-PAGE to separate individual subunits

• Detect with specific antibodies against each subunit

Proximity ligation assay:

• Use antibodies against different ENaC subunits from different species

• Apply species-specific secondary antibodies conjugated to oligonucleotides

• When antibodies are in close proximity (<40 nm), oligonucleotides can be ligated

• Amplify signal through rolling circle amplification

• Detect with fluorescent probes

Recent structural studies, as seen in search result , have revealed that ENaC can form various heteromeric assemblies. The γ subunit (SCNN1G) consistently occupies position 3 in these assemblies, while δ or α takes position 1, and β occupies position 2 .

What strategies can address the challenges of detecting SCNN1G in tissues with low expression levels?

Several advanced approaches can enhance detection of low-abundance SCNN1G:

Signal amplification methods:

• Tyramide signal amplification (TSA):

  • After primary and HRP-conjugated secondary antibody incubation

  • Apply tyramide solution which deposits multiple fluorophores

  • Can increase sensitivity 10-100 fold over conventional methods

• Polymer-based detection systems:

  • Use polymers conjugated with multiple secondary antibodies and HRP

  • Provides significant signal enhancement without background increase

• RNAscope® with immunofluorescence:

  • Combine mRNA detection with protein immunostaining

  • Validate protein expression with transcript detection

  • Useful when protein expression is below antibody detection threshold

Sample enrichment techniques:

• Laser capture microdissection:

  • Isolate epithelial cells from complex tissues

  • Concentrate target cells before protein extraction

• Cell surface protein biotinylation:

  • Selectively label and purify membrane proteins

  • Enrich for SCNN1G before detection

• Subcellular fractionation:

  • Isolate membrane fractions to concentrate channels

  • Reduce background from cytoplasmic proteins

These approaches can significantly improve detection sensitivity for challenging samples, though each requires careful optimization and appropriate controls .

How can researchers investigate the functional relationship between SCNN1G expression and channel activity?

Comprehensive investigation requires combining molecular and electrophysiological techniques:

Patch-clamp electrophysiology:

• Whole-cell configuration to measure amiloride-sensitive currents

• Single-channel recordings to analyze channel kinetics

• Correlate current measurements with SCNN1G expression levels

• Test response to known modulators (amiloride at 50-100 nM, zinc, proteases)

Fluorescence-based assays:

• Sodium-sensitive fluorescent indicators (SBFI, CoroNa Green)

• Live cell imaging to monitor sodium influx

• Correlate with SCNN1G immunofluorescence in fixed cells

Combined approaches:

• siRNA or CRISPR knockout of SCNN1G followed by functional assays

• Rescue experiments with wild-type vs. mutant SCNN1G expression

• Correlation of protein expression with electrophysiological parameters

The research by Schreiber et al. demonstrates how SCNN1G expression correlates with functional ENaC activity in ASCL1-dependent small cell lung cancer. They showed that pharmacological inhibition of ENaC with amiloride had stronger growth inhibition effects in ASCL1-dependent SCLC compared to ASCL1-independent SCLC, establishing a direct functional link between SCNN1G expression and biological outcomes .

What are common challenges in SCNN1G antibody applications and how can they be addressed?

Researchers frequently encounter these issues when working with SCNN1G antibodies:

Specific optimization approaches:

• For signal enhancement in IHC: Test both TE buffer (pH 9.0) and citrate buffer (pH 6.0) for antigen retrieval

• For membrane protein detection: Include membrane solubilization steps with appropriate detergents

• For glycoprotein analysis: Compare samples with and without glycosidase treatment

• For specificity verification: Include peptide competition controls with the immunizing peptide

How can researchers validate SCNN1G antibody specificity for their experimental system?

Comprehensive validation should include multiple complementary approaches:

Genetic validation:

• siRNA/shRNA knockdown:

  • Transfect cells with SCNN1G-targeting siRNA/shRNA

  • Compare antibody signal between knockdown and control cells

  • Signal should be significantly reduced in knockdown samples

  • Similar to methods used by Schreiber et al. where ASCL1 suppression reduced SCNN1G expression

• CRISPR/Cas9 knockout:

  • Generate SCNN1G knockout cell lines

  • Verify complete absence of signal in knockout cells

  • Include wild-type cells as positive control

Biochemical validation:

• Peptide competition:

  • Pre-incubate antibody with excess immunizing peptide

  • Apply to parallel samples

  • Specific signal should be abolished or significantly reduced

  • As demonstrated in immunostaining experiments where peptide competition greatly reduced detection

• Overexpression systems:

  • Transfect cells with tagged SCNN1G constructs

  • Compare detection with anti-tag and anti-SCNN1G antibodies

  • Signals should overlay in co-localization studies

• Multiple antibody validation:

  • Test different antibodies targeting distinct epitopes

  • Consistent patterns across antibodies increase confidence

  • Compare commercially available antibodies (e.g., 13943-1-AP and 30037-1-AP)

Orthogonal validation:

• Transcript-protein correlation:

  • Perform qRT-PCR for SCNN1G mRNA

  • Compare with protein levels across samples

  • Transcript and protein levels should correlate

• Mass spectrometry validation:

  • Immunoprecipitate SCNN1G and analyze by mass spectrometry

  • Confirm peptide sequences match target protein

What approaches can address the challenge of detecting SCNN1G in heteromeric complexes with other ENaC subunits?

Detecting SCNN1G within native heteromeric complexes requires specialized approaches:

Crosslinking strategies:

• Chemical crosslinking:

  • Apply membrane-permeable crosslinkers (DSS, BS3)

  • Stabilize protein-protein interactions before cell lysis

  • Analyze complexes by SDS-PAGE and Western blotting

  • Identify SCNN1G-containing complexes at higher molecular weights

• Photoactivatable crosslinkers:

  • More specific spatial control for crosslinking

  • Useful for capturing transient interactions

Affinity purification of intact complexes:

• Tandem affinity purification:

  • Express tagged version of one ENaC subunit

  • Purify intact complexes under native conditions

  • Detect SCNN1G in purified material

• Immunoaffinity chromatography:

  • Immobilize anti-SCNN1G antibodies on solid support

  • Purify native complexes from solubilized membranes

  • Identify interacting partners by Western blot or mass spectrometry

Structural biology approaches:

• Fluorescence-detection size-exclusion chromatography (FSEC):

  • As used in the structural study by Martin et al.

  • Monitor heteromeric complexes by tagging one subunit (e.g., eGFP-fused γ subunit)

  • Assess complex formation and stability

• Blue native PAGE followed by immunoblotting:

  • Preserve native complexes during electrophoresis

  • Detect SCNN1G in higher molecular weight complexes

  • Second dimension SDS-PAGE can reveal subunit composition

Recent structural studies have provided important insights into how SCNN1G assembles with other ENaC subunits, revealing that the γ subunit consistently occupies position 3 in heteromeric complexes .

How can SCNN1G antibodies be used to investigate Liddle syndrome and other ENaC-related disorders?

SCNN1G antibodies provide valuable tools for investigating ENaC-related disorders:

For Liddle syndrome research:

• Mutation-specific approaches:

  • Generate antibodies against common mutant regions (e.g., p.Pro625Leu)

  • Compare wild-type vs. mutant SCNN1G expression and localization

  • Study described in Xu et al. identified a novel SCNN1G missense variant (c.1874C>T, p.Pro625Leu) in pediatric Liddle syndrome

• Cell surface expression analysis:

  • Surface biotinylation assays to quantify membrane expression

  • Compare surface/total SCNN1G ratios between normal and patient samples

  • Increased surface expression is characteristic of Liddle syndrome mutations

• Degradation pathway investigation:

  • Pulse-chase experiments with cycloheximide to measure protein half-life

  • Assess ubiquitination status using anti-ubiquitin co-immunoprecipitation

  • Liddle syndrome mutations typically affect degradation pathways, leading to increased channel residence time at the cell surface

For pseudohypoaldosteronism research:

• Tissue expression profiling:

  • Compare SCNN1G expression in normal vs. patient samples

  • Analyze co-expression with other ENaC subunits

  • Loss-of-function mutations in alpha- or beta-ENaC cause pseudohypoaldosteronism (PHA-1)

• Functional correlation studies:

  • Combine immunostaining with electrophysiological measurements

  • Assess correlation between protein expression and channel function

  • Identify compensatory mechanisms in disease states

What methodological approaches can researchers use to study the role of SCNN1G in cancer biology?

The role of SCNN1G in cancer can be investigated through multiple complementary approaches:

Expression analysis in cancer tissues:

• Tissue microarray immunohistochemistry:

  • Compare SCNN1G expression across tumor types and stages

  • Correlate with clinical outcomes

  • Include matched normal adjacent tissue controls

• Single-cell analysis:

  • Combined immunofluorescence with other cancer markers

  • Assess heterogeneity of expression within tumors

  • Identify specific cancer cell populations expressing SCNN1G

Functional studies in cancer models:

• Pharmacological approach:

  • Treat cancer cells with ENaC inhibitors (amiloride)

  • Measure effects on proliferation, migration, and survival

  • Compare sensitivity between SCNN1G-expressing and non-expressing cancers

  • As demonstrated by Schreiber et al. where amiloride inhibited growth of ASCL1-dependent SCLC more strongly than ASCL1-independent SCLC

• Genetic manipulation:

  • Knockdown or overexpress SCNN1G in cancer cell lines

  • Assess effects on cancer hallmarks (proliferation, invasion, etc.)

  • Evaluate downstream signaling pathways

• Patient-derived xenograft models:

  • Establish PDX models from SCNN1G-expressing tumors

  • Test therapeutic targeting approaches

  • Correlate SCNN1G expression with treatment response

Mechanistic investigations:

• Interactome analysis:

  • Identify cancer-specific interaction partners through IP-MS

  • Map SCNN1G-dependent signaling networks

  • Compare interactomes between normal and cancer cells

• Sodium imaging in live cancer cells:

  • Monitor intracellular sodium using fluorescent indicators

  • Correlate with SCNN1G expression

  • Investigate how altered sodium homeostasis affects cancer cell biology

The research by Schreiber et al. provides an excellent framework, demonstrating that SCNN1A/αENaC is a direct transcriptional target of the neuroendocrine lung cancer lineage oncogene ASCL1 that can be pharmacologically targeted with antitumor effects .

How can researchers utilize SCNN1G antibodies to investigate the regulation of epithelial sodium transport in different physiological conditions?

SCNN1G antibodies can illuminate regulatory mechanisms across various physiological states:

Hormonal regulation studies:

• Aldosterone response:

  • Treat cells/tissues with aldosterone in time-course experiments

  • Monitor SCNN1G expression, localization, and post-translational modifications

  • Correlate with electrophysiological measurements of sodium transport

• Stress hormone effects:

  • Examine SCNN1G regulation under glucocorticoid treatment

  • Compare with mineralocorticoid-specific effects

  • Assess tissue-specific responses

Physiological adaptation models:

• Salt restriction/loading protocols:

  • Subject animal models to controlled dietary interventions

  • Analyze SCNN1G expression and localization in kidney tubules

  • Correlate with sodium excretion measurements

• Hypertension models:

  • Compare SCNN1G expression in normotensive vs. hypertensive animals

  • Evaluate effects of antihypertensive treatments on SCNN1G

  • Investigate cell surface expression and turnover rates

Pathophysiological conditions:

• Inflammatory states:

  • Analyze SCNN1G regulation during cytokine exposure

  • Compare expression in normal vs. inflamed tissues

  • Investigate mechanisms linking inflammation to sodium transport

• Hypoxia response:

  • Examine SCNN1G expression under normoxic vs. hypoxic conditions

  • Correlate with HIF-1α activity

  • Particularly relevant in lung and cancer research

Advanced methodological approaches:

• In vivo proximity labeling:

  • Express SCNN1G fused to promiscuous biotin ligases (BioID, TurboID)

  • Identify proteins in spatial proximity under different conditions

  • Map dynamic regulation of the channel's microenvironment

• Super-resolution microscopy:

  • Track SCNN1G localization at nanoscale resolution

  • Monitor clustering and diffusion dynamics

  • Assess co-localization with regulatory proteins

• Quantitative phospho-proteomics:

  • Immunoprecipitate SCNN1G under different conditions

  • Analyze phosphorylation status by mass spectrometry

  • Identify regulatory kinases and phosphatases