SCN4B Antibody, HRP conjugated

What is SCN4B and why is it an important research target?

SCN4B encodes the β4 subunit of voltage-gated sodium channels, initially characterized in excitable tissues but now known to be expressed in epithelial cells. Research has demonstrated it functions as a metastasis-suppressor gene in breast cancer, with reduced expression levels correlating with high-grade primary and metastatic tumors . SCN4B overexpression has been shown to suppress cancer cell invasiveness and tumor progression, making it a valuable target for oncology research .

What specific epitope does the HRP-conjugated SCN4B antibody recognize?

The HRP-conjugated SCN4B antibody typically recognizes amino acids 31-162 of the human SCN4B protein . This region is important for the protein's functional activity and provides high specificity for detection in experimental applications. The epitope selection ensures minimal cross-reactivity with other sodium channel subunits.

What applications is the HRP-conjugated SCN4B antibody validated for?

The HRP-conjugated SCN4B antibody has been validated for Western blotting (WB) and ELISA applications . The direct HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal. Some non-conjugated versions of SCN4B antibodies have also been validated for immunohistochemistry (IHC) applications .

What is the expected molecular weight when detecting SCN4B in Western blot applications?

While the calculated molecular weight of SCN4B is approximately 25 kDa, the observed molecular weight in Western blot applications is typically around 39 kDa . This discrepancy likely reflects post-translational modifications such as glycosylation, which are common for membrane-associated proteins like sodium channel subunits.

What are the optimal dilution ratios for different applications of SCN4B HRP-conjugated antibody?

Based on validated protocols, the recommended dilution ranges are:

• Western Blotting: 1:500-1:2000

• ELISA: 1:2000-1:10000

These ranges provide starting points for optimization in specific experimental systems. Researchers should perform dilution series experiments to determine the optimal concentration for their particular application and sample type.

What sample preparation methods are recommended for optimal SCN4B detection?

For Western blotting applications, cells or tissues should be lysed in a buffer containing protease inhibitors to prevent degradation of the target protein. For immunofluorescence applications with non-HRP conjugated SCN4B antibodies, samples are typically fixed in 4% formaldehyde and permeabilized with 0.2% Triton X-100 . These preparation methods help preserve protein integrity while enabling efficient antibody access to the target epitope.

How should the SCN4B HRP-conjugated antibody be stored to maintain optimal activity?

For long-term storage (up to one year), the antibody should be kept at -20°C . For frequent use over shorter periods (up to one month), 4°C storage is appropriate. The antibody is typically stored in a buffer containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide to maintain stability . Repeated freeze-thaw cycles should be strictly avoided as they can compromise antibody performance.

What blocking conditions are most effective when using SCN4B HRP-conjugated antibodies?

For Western blotting applications, 5% BSA or non-fat dry milk in TBST is commonly effective. For immunofluorescence applications, 10% normal goat serum has been validated as an effective blocking agent . The optimal blocking solution may need to be determined empirically based on the specific tissue or cell type being analyzed.

How can the SCN4B antibody be used to investigate its role in cancer progression?

Research has shown that SCN4B acts as a metastasis-suppressor gene, with reduced expression correlating with increased cancer aggressiveness . Researchers can use SCN4B antibodies to:

• Compare expression levels between normal epithelial cells, primary tumors, and metastatic lesions

• Examine correlations between SCN4B expression and clinical parameters

• Validate functional studies where SCN4B has been experimentally modulated

• Explore co-localization with markers of cell migration and invasion

These approaches can provide insights into how SCN4B downregulation promotes the acquisition of an amoeboid-mesenchymal hybrid phenotype in cancer cells .

How can SCN4B antibodies contribute to understanding its role in the cGMP-PKG pathway?

Recent research has demonstrated that SCN4B overexpression suppresses the expression of PKG1 and the phosphorylation of downstream proteins in non-small cell lung cancer (NSCLC) . SCN4B antibodies can be used in combination with antibodies against components of the cGMP-PKG pathway to:

• Validate expression changes following experimental modulation of SCN4B

• Examine co-localization patterns in tissue samples

• Develop predictive biomarker panels for NSCLC progression

• Investigate potential therapeutic interventions targeting this pathway

What approaches can be used to distinguish between SCN4B-dependent and sodium channel-independent functions?

Research has shown that SCN4B's role in inhibiting cancer cell invasion is independent of its function as an auxiliary subunit of voltage-gated sodium channels . To investigate these independent functions, researchers can:

• Use SCN4B antibodies in cells where sodium channels have been knocked down

• Compare the effects of SCN4B modulation in the presence of sodium channel blockers like TTX

• Analyze the localization of SCN4B in relation to sodium channel alpha subunits

• Perform domain-specific functional studies using the C-terminus of β4, which has been shown to prevent hyperactivated migration

What are common causes of non-specific binding when using SCN4B HRP-conjugated antibodies?

Non-specific binding can result from several factors:

• Insufficient blocking of non-specific binding sites

• Too high antibody concentration

• Inadequate washing steps

• Sample-specific factors (high lipid content, unusual protein composition)

• Cross-reactivity with structurally similar proteins

To minimize non-specific binding, researchers should optimize blocking conditions, antibody dilutions, and washing protocols. Including appropriate negative controls (samples known to lack SCN4B expression) can help identify non-specific signals.

How can researchers validate the specificity of SCN4B antibody signals?

To ensure signal specificity, researchers should consider multiple validation approaches:

• Comparing results with alternative antibodies targeting different SCN4B epitopes

• Using positive and negative control samples with known SCN4B expression status

• Performing siRNA/shRNA knockdown experiments to confirm signal reduction

• Testing for signal absence in SCN4B knockout models when available

• Verifying the molecular weight of detected bands in Western blot applications

In published research, siRNA knockdown of SCN4B has been shown to validate antibody specificity while also providing functional insights .

How should researchers interpret discrepancies between SCN4B protein expression and functional data?

When interpreting discrepancies, researchers should consider:

• Post-translational modifications affecting protein activity without changing total protein levels

• Context-dependent protein interactions that modulate function

• Subcellular localization changes that affect protein activity

• Threshold effects where small expression changes lead to significant functional outcomes

• Temporal dynamics of protein expression versus functional responses

Research has shown that even partial reduction of SCN4B expression can significantly increase RhoA activity and enhance cancer cell invasiveness, indicating potential threshold effects .

How can SCN4B antibodies be used to investigate epithelial-to-mesenchymal transition (EMT) in cancer?

SCN4B downregulation has been linked to increased cancer cell migration and acquisition of an amoeboid-mesenchymal hybrid phenotype . Researchers can use SCN4B antibodies to:

• Monitor changes in SCN4B expression during EMT induction

• Correlate SCN4B levels with established EMT markers

• Investigate the relationship between SCN4B expression and RhoA activity during EMT

• Examine how SCN4B restoration affects the mesenchymal phenotype of cancer cells

What considerations are important when developing multiplex assays including SCN4B detection?

When developing multiplex assays, researchers should consider:

• Antibody compatibility (host species, isotype, detection system)

• Potential cross-reactivity between multiple antibodies

• Signal strength balance between different targets

• Optimization of fixation and permeabilization protocols that work for all targets

• Sequential detection strategies when direct multiplexing is challenging

For studies examining SCN4B in relation to the cGMP-PKG pathway, careful selection of compatible antibodies against PKG1 and its downstream targets is essential .

Table 1: Properties of SCN4B Antibody, HRP Conjugated

Table 2: Recommended Experimental Conditions for SCN4B Antibody Applications