SHOC2 Antibody, Biotin conjugated

What is SHOC2 and why is it a significant target for antibody development?

SHOC2 (also known as Sur8) is a scaffold protein crucial in the ERK1/2 signaling pathway. It's primarily composed of leucine-rich repeats (LRR) with a lysine-rich sequence at the amino terminus. SHOC2 acts as a positive modulator of the RAS-MAPK signaling cascade and, together with protein phosphatase 1c (PP1c), forms a highly specific M-Ras effector complex essential for activation of the MAPK pathway by growth factors .

The significance of SHOC2 as a research target is underscored by its involvement in:

• Cancer biology and tumorigenesis

• Noonan-like syndrome with loose anagen hair (NSLAH), a developmental disorder caused by mutations in the SHOC2 gene

• Regulation of the ERK1/2 pathway, which is critical in organismal development and tissue morphogenesis

How does biotin conjugation enhance SHOC2 antibody functionality?

Biotin conjugation significantly enhances antibody functionality through:

• High-affinity interaction: Biotin forms a stable, non-covalent interaction with Streptavidin/Avidin, creating a strong and highly specific bond

• Signal amplification: Multiple biotin molecules (>4) can be attached to each antibody, and each streptavidin molecule can bind four biotin molecules, creating a tetravalent binding mode that amplifies detection signals

• Versatility: Biotin-conjugated antibodies can be used with various detection systems by employing streptavidin conjugated to fluorescent dyes or reporter enzymes such as HRP or AP

• Sensitivity: The multivalent properties of the biotin-streptavidin system enable robust detection of low abundance targets

What are the recommended storage conditions for SHOC2 biotin-conjugated antibodies?

For optimal performance and stability, SHOC2 biotin-conjugated antibodies should be stored according to these guidelines:

• Long-term storage: Store at -20°C for up to one year from the date of receipt

• After reconstitution: Can be stored at 4°C for one month

• Aliquoting: For extended use, aliquot and store at -20°C for up to six months

• Avoid freeze-thaw cycles: Repeated freezing and thawing should be minimized as this can degrade the antibody and reduce its efficacy

How can SHOC2 biotin-conjugated antibodies be used effectively in protein-protein interaction studies?

SHOC2 biotin-conjugated antibodies are valuable tools for investigating protein-protein interactions within the SHOC2 scaffolding complex. Based on published research methodologies:

Recommended protocol:

• Immunoprecipitation setup:

  • Lyse cells in RIPA buffer on ice for 30 minutes

  • Clear lysates by centrifugation

  • Incubate 500 μg of cell lysate with biotin-conjugated SHOC2 antibody (2-5 μg)

• Complex isolation:

  • Use streptavidin agarose columns to pull down the biotin-antibody-protein complexes

  • Wash thoroughly to remove non-specific binding

  • Elute bound proteins under appropriate conditions

• Analysis of binding partners:

  • Analyze by Western blotting using antibodies against suspected SHOC2 interacting proteins

  • Known interaction partners include RAF-1, M-RAS, PP1c, HUWE1, USP7, and VCP/PSMC5

This approach has successfully identified multiple SHOC2 binding partners, as demonstrated in studies where high-affinity SHOC2 antibodies efficiently immunoprecipitated several known SHOC2 interacting partners .

What optimization strategies should be employed when using SHOC2 biotin-conjugated antibodies in immunofluorescence studies?

For optimal results in immunofluorescence applications with SHOC2 biotin-conjugated antibodies:

Sample preparation:

• Fix cells using 4% paraformaldehyde

• Permeabilize with appropriate buffer (e.g., 0.1% Triton X-100)

Antibody concentration optimization:

• Initial concentration: 2 μg/ml for paraffin-embedded sections

• Titrate to determine optimal concentration for your specific sample

Antigen retrieval:

• For paraffin-embedded sections: Heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

• Block with 10% goat serum to reduce background

Detection system:

• Use streptavidin conjugated to appropriate fluorophores

• For multiplex imaging, select fluorophores with minimal spectral overlap

• Include controls to assess autofluorescence and non-specific binding

Image acquisition:

• Use appropriate filter sets for the selected fluorophores

• Acquire images using consistent exposure settings

• Consider using a system like the Mariannas Imaging system with a cooled CCD for optimal results

What are the critical parameters for using SHOC2 biotin-conjugated antibodies in Western blot applications?

For optimal Western blot results with SHOC2 biotin-conjugated antibodies:

Sample preparation:

• Load 30 μg of protein per lane under reducing conditions

• Use 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving) for 2-3 hours

Protein transfer:

• Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

Blocking and antibody incubation:

• Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Optimal antibody concentration: 0.5-2 μg/mL

• Incubate overnight at 4°C

Washing and detection:

• Wash with TBS-0.1% Tween 3 times (5 minutes each)

• For detection, use streptavidin-HRP and develop with ECL detection system

• Expected band size for SHOC2: approximately 70 kDa

How can SHOC2 biotin-conjugated antibodies be employed to study ERK1/2 pathway regulation and dysregulation?

SHOC2 biotin-conjugated antibodies can be powerful tools for investigating ERK1/2 pathway regulation:

Experimental approach:

• Pathway stimulation:

  • Stimulate cells with appropriate growth factors (e.g., EGF)

  • Monitor ERK1/2 phosphorylation levels as readout of pathway activation

• Biotin-antibody complex isolation:

  • Use biotin-conjugated SHOC2 antibodies to pull down the SHOC2 complex at different time points following stimulation

  • Analyze complex composition changes during pathway activation/deactivation

• Comparative analysis:

  • Compare wildtype SHOC2 with disease-associated variants (e.g., S2G mutation in NSLAH)

  • Analyze differences in complex formation and ERK1/2 activation

Research findings example:
Studies have demonstrated that intracellular expression of high-affinity SHOC2 antibodies (as intrabodies) can alter ERK1/2 phosphorylation levels. Specifically, cells expressing high-affinity antibodies targeting the hinge region between the N-terminus and LRR domain of SHOC2 showed increased phospho-ERK1/2 levels .

What strategies can be used to validate the specificity of SHOC2 biotin-conjugated antibodies?

Rigorous validation of SHOC2 biotin-conjugated antibodies should include:

Multiple validation approaches:

• Immunoprecipitation followed by mass spectrometry:

  • Use biotin-conjugated SHOC2 antibodies to pull down proteins

  • Analyze by mass spectrometry to confirm SHOC2 as the primary target

• SHOC2 knockdown/knockout validation:

  • Compare antibody signal in wildtype cells versus SHOC2 knockdown cells

  • The SHOC2 siRNA sequence 5′-AUACAGAUGUACGAGUCCATT-3′ has been validated for efficient knockdown

  • Use CRISPR knockout SHOC2 HeLa cells as a definitive negative control

• Epitope mapping:

  • Use a series of SHOC2 deletion mutants to determine specific binding regions

  • This approach has successfully mapped antibody binding regions in previous studies

• Binding kinetics characterization:

  • Use bio-layer interferometry to determine binding affinity

  • High-specificity antibodies typically show affinity in the nanomolar range (e.g., Kd values 14.4 nM (±0.4) to 516 nM (±12))

How can researchers develop and optimize Single-Domain Antibodies (sdAbs) against SHOC2 for advanced applications?

Development of high-quality SHOC2-specific sdAbs involves:

Selection and production protocol:

• Library screening:

  • Screen phage display library (3×10^9 hs2dAb clones) using biotinylated SHOC2

  • Create a yeast two-hybrid (Y2H) library with positive clones (complexity ~1.19×10^6 cells)

  • Use full-length SHOC2 as bait in Y2H analysis

• Reformatting selected sdAbs:

  • Sub-clone into eukaryotic expression vector with C-terminal chimeric rabbit-Fc-IgG2

  • Alternatively, create mCherry-tagged sdAbs for intracellular expression and visualization

• Expression and purification:

  • Express in HEK293 cells in serum-free media for 4-6 days

  • Harvest culture supernatants containing secreted sdAbs (~40 kDa)

• Validation of binding specificity:

  • Test immunoprecipitation of both ectopically expressed SHOC2-tRFP and endogenous SHOC2

  • Determine binding kinetics using bio-layer interferometry

  • Map binding epitopes using SHOC2 deletion mutants

Performance data from published research:
High-quality SHOC2 sdAbs have demonstrated Kd values in the nanomolar range, with hs2dAb B99 showing particularly tight interaction (Kd = 14.4 nM ±0.4) .

How can SHOC2 biotin-conjugated antibodies facilitate research on mutations associated with Noonan-like syndrome?

SHOC2 biotin-conjugated antibodies provide valuable tools for investigating NSLAH-associated mutations:

Research approaches:

• Comparative analysis of wildtype vs. mutant SHOC2:

  • Express wildtype SHOC2 and disease-associated variants (S2G, E89D, C238Y, L473I) in SHOC2 knockout cells

  • Use biotin-conjugated antibodies to immunoprecipitate SHOC2 complexes

  • Compare binding partners and complex composition

• Ubiquitylation analysis:

  • NSLAH-associated SHOC2 variants show aberrant ubiquitylation patterns

  • Use denaturing conditions to immunoprecipitate SHOC2 and analyze ubiquitylation levels

• Intracellular localization studies:

  • The S2G mutation causes aberrant N-myristoylation and membrane localization

  • Use immunofluorescence to track subcellular localization differences

• Therapeutic exploration:

  • Test compounds that may rescue function (e.g., 2-hydroxymyristic acid, an NMT inhibitor, reduces aberrant membrane localization of SHOC2 S2G)

  • Some antibodies have shown partial rescue of ERK1/2 phosphorylation in cells carrying SHOC2-S2G mutations

What are effective strategies for reducing background when using SHOC2 biotin-conjugated antibodies?

High background is a common challenge with biotin-conjugated antibodies. To minimize this issue:

Optimization strategies:

• Block endogenous biotin:

  • Pre-block endogenous biotin with avidin/streptavidin followed by free biotin

  • Use commercial endogenous biotin-blocking kits prior to primary antibody incubation

• Optimize blocking conditions:

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Add 0.1-0.3% Triton X-100 to blocking buffer for permeabilization

  • Consider using commercial protein-free blockers if serum blocking is insufficient

• Antibody dilution optimization:

  • Titrate antibody concentration (typical range: 0.5-2 μg/ml)

  • Increase wash stringency (number of washes and detergent concentration)

• Detection system considerations:

  • For low abundance targets, HRP or AP enzyme-based systems may provide better signal-to-noise ratio than direct fluorescence

  • Consider using tyramide signal amplification for maximum sensitivity while maintaining low background

• Tissue/sample-specific approaches:

  • For tissues with high endogenous biotin (e.g., liver, kidney), consider using non-biotin detection methods

  • For fixed tissues, ensure complete quenching of fixative (e.g., with glycine or ammonium chloride)

How can researchers troubleshoot failed detection with SHOC2 biotin-conjugated antibodies?

When SHOC2 detection fails despite using biotin-conjugated antibodies, consider this systematic troubleshooting approach:

Methodical troubleshooting process:

• Antibody validation:

  • Confirm antibody activity using positive control samples (e.g., Jurkat, MCF-7, HepG2 cell lysates)

  • SHOC2 is widely expressed but expression levels vary between cell lines

• Sample preparation issues:

  • For immunohistochemistry: Ensure proper antigen retrieval using EDTA buffer (pH 8.0)

  • For Western blot: Verify complete protein denaturation and efficient transfer

• Epitope accessibility:

  • SHOC2 antibodies may target conformational epitopes that are sensitive to denaturation

  • Some SHOC2 antibodies recognize conformational epitopes and won't work for Western blotting

• Detection system problems:

  • Verify streptavidin-conjugate activity with a control biotin-antibody

  • Check for degradation of detection reagents (fluorophores, enzymes)

  • Ensure secondary detection system is compatible with the biotin-conjugated primary antibody

• Signal development issues:

  • For chemiluminescent detection: Extend exposure time incrementally

  • For immunofluorescence: Adjust gain/exposure settings and use appropriate filters

What are the critical considerations when designing experiments to detect protein interactions using SHOC2 biotin-conjugated antibodies?

For successful protein interaction studies with SHOC2 biotin-conjugated antibodies:

Experimental design considerations:

• Lysis conditions:

  • Use mild lysis conditions to preserve protein-protein interactions

  • RIPA buffer has been validated for SHOC2 complex isolation

  • Include protease and phosphatase inhibitors to preserve post-translational modifications

• Biotin conjugation ratio:

  • Optimal biotin:antibody ratio is critical - excessive biotinylation can disrupt antigen binding

  • Commercial conjugation kits (e.g., LYNX Rapid Plus) provide consistent conjugation efficiency

• Binding competition:

  • Be aware that some SHOC2 antibodies may compete with interacting proteins if they target the same epitope

  • Use epitope-mapped antibodies to avoid disrupting important protein interfaces

• Control experiments:

  • Include IgG control to identify non-specific binding

  • Use SHOC2-depleted cell lysates as negative controls

  • Consider pre-clearing lysates with protein A/G beads to reduce background

• Verification approaches:

  • Confirm interactions using reciprocal immunoprecipitation

  • For novel interactions, verify with orthogonal methods (e.g., proximity ligation assay, FRET)

  • Known SHOC2 interactors (M-RAS, RAF-1, PP1c, HUWE1, USP7) can serve as positive controls

How can cleavable biotin-conjugated SHOC2 antibodies be developed for advanced purification applications?

Cleavable biotin-conjugated SHOC2 antibodies represent an advanced tool for purification applications:

Development methodology:

• Cleavable linker chemistry:

  • Incorporate a disulfide bond in the biotin-antibody linker

  • This allows elution of captured proteins under mild reducing conditions without disrupting antigen-antibody binding

• Synthesis approach:

  • Couple SHOC2 antibodies with biotin derivatives containing a disulfide bond

  • Verify that the conjugate can simultaneously bind to anti-SHOC2 antibody and NeutrAvidin

  • Confirm efficient cleavage with dithiothreitol (DTT) treatment

• Application in protein complex isolation:

  • Immobilize biotin-conjugated SHOC2 antibodies on solid phase via NeutrAvidin

  • Incubate with cell lysates expressing SHOC2 and its binding partners

  • Wash thoroughly to remove non-specific binding

  • Elute specifically bound proteins with mild DTT treatment

  • This approach maintains native protein conformation and preserves complexes for downstream analysis

How can SHOC2 biotin-conjugated antibodies be used to identify novel therapeutic targets in RAS-MAPK pathway-driven cancers?

SHOC2 biotin-conjugated antibodies offer unique opportunities for drug discovery in RAS-MAPK pathway-driven cancers:

Strategic research approaches:

• Target identification using chemical proteomics:

  • Use biotin-conjugated SHOC2 antibodies to pull down the SHOC2 complex

  • Combine with small molecule inhibitors (e.g., Celastrol) to identify changes in complex composition

  • Analyze by mass spectrometry to identify proteins that dissociate from the complex upon treatment

• Scaffold-focused drug screening:

  • Use biotin-conjugated SHOC2 antibodies in high-throughput pull-down assays

  • Screen for compounds that disrupt specific protein-protein interactions within the SHOC2 scaffold

  • Target the SHOC2-PP1c holoenzyme as a potential therapeutic target

• Validation in cancer models:

  • In tumor cells with RAS gene mutations, inhibition of SHOC2 expression inhibits MAPK but not PI3K activity

  • Use biotin-conjugated SHOC2 antibodies to track complex composition changes in response to potential therapeutics

Research finding example:
Celastrol has been identified as a compound that binds to SHOC2, and this binding can be detected using biotin-Celastrol conjugates in pull-down assays with streptavidin agarose columns. Analysis of the pulled-down material by Western blotting confirmed SHOC2 as a target of Celastrol .

What are the latest developments in using SHOC2 biotin-conjugated antibodies for intracellular delivery of therapeutic agents?

Emerging research explores SHOC2 biotin-conjugated antibodies for targeted delivery of therapeutics:

Advanced applications:

• Intrabody development:

  • Express SHOC2 antibodies intracellularly as mCherry-tagged constructs

  • These intrabodies can be used to visualize endogenous SHOC2 and modulate its function

  • High-affinity antibodies targeting the hinge region between the N-terminus and LRR domain of SHOC2 have shown promising results

• Therapeutic potential:

  • Research has demonstrated that certain SHOC2 antibodies can alter ERK1/2 signaling when expressed intracellularly

  • Some antibodies have partially rescued ERK1/2 phosphorylation in cells carrying the SHOC2-S2G-tRFP mutant

• Delivery strategies for antibody-based therapeutics:

  • Biotin-streptavidin bridging can be used to create multimodal therapeutic complexes

  • Biotin-conjugated SHOC2 antibodies can be coupled with streptavidin-conjugated therapeutic moieties

  • This approach allows for the targeted delivery of siRNAs, drug payloads, or imaging agents to cells with aberrant SHOC2 signaling