SHOC2 Antibody, HRP conjugated

What is SHOC2 and why is it important to study?

SHOC2 is a leucine-rich repeat protein that functions as a regulatory subunit of protein phosphatase 1 (PP1c). It acts as an M-Ras/MRAS effector and participates in MAPK pathway activation. Upon M-Ras/MRAS activation, SHOC2 targets PP1c to specifically dephosphorylate the 'Ser-259' inhibitory site of RAF1 kinase and stimulate RAF1 activity at specialized signaling complexes . SHOC2 plays a vital role in transformation, metastasis, epithelial-to-mesenchymal transition, and MAPK pathway inhibitor resistance, making it an important subject of study in cancer research . Additionally, defects in SHOC2 are associated with Noonan syndrome-like with loose anagen hair (NSLAH), characterized by macrocephaly, dysmorphic facial features, and hair abnormalities .

What are the optimal applications for SHOC2 Antibody, HRP Conjugated?

The SHOC2 Antibody, HRP Conjugated is optimized for several applications with specific recommended dilutions:

For optimal results in Western blotting, the antibody can detect SHOC2 protein in various human, mouse, and rat tissues where it localizes primarily in the cytoplasm and nucleus . The HRP conjugation eliminates the need for secondary antibody incubation, streamlining experimental workflows and potentially reducing background signal.

How should SHOC2 Antibody, HRP Conjugated be stored and handled for maximum stability?

For maximum stability and retention of activity, SHOC2 Antibody, HRP Conjugated should be stored at -20°C . The antibody comes in an aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol . To prevent activity loss from repeated freeze-thaw cycles, it is recommended to aliquot the antibody into multiple smaller volumes before freezing . When handling, avoid contamination and minimize exposure to light and heat which can diminish HRP enzymatic activity. Prior to use, allow the antibody to equilibrate to room temperature and gently mix by inversion rather than vortexing, which can damage the antibody.

What controls should be included when using SHOC2 Antibody, HRP Conjugated?

When designing experiments with SHOC2 Antibody, HRP Conjugated, several controls should be included to ensure valid and interpretable results:

• Positive control: Lysates from cells known to express SHOC2 (such as HeLa or A549 cell lines)

• Negative control: Lysates from cells where SHOC2 has been knocked down via siRNA or CRISPR

• Loading control: Antibody against a housekeeping protein (such as β-actin or GAPDH) to ensure equal loading of samples

• HRP activity control: Including an HRP substrate-only well to check for any non-specific signal

• Peptide competition control: Pre-incubating the antibody with its immunizing peptide to confirm specificity

For immunohistochemistry applications, additional tissue-specific positive and negative controls should be included, along with a primary antibody omission control to assess potential background staining.

How can SHOC2 Antibody be used to investigate the SHOC2-MRAS-PP1C (SMP) complex formation?

The SHOC2-MRAS-PP1C (SMP) complex represents a critical regulatory assembly in the MAPK signaling pathway. To investigate this complex using SHOC2 Antibody, HRP conjugated, researchers can employ co-immunoprecipitation (co-IP) followed by Western blotting. The experimental approach would involve:

• Preparing cell lysates from cells in different activation states (serum-starved vs. growth factor stimulated)

• Performing co-IP using either anti-SHOC2, anti-MRAS, or anti-PP1C antibodies

• Analyzing the immunoprecipitates by Western blotting using the SHOC2 Antibody, HRP conjugated (1:1000 dilution)

It's important to note that the SMP complex formation is dependent on MRAS being in the GTP-bound active state . Therefore, researchers should include conditions that activate MRAS (such as EGF stimulation) and compare with inactive conditions. Additionally, researchers could use GMPPNP (a non-hydrolyzable GTP analog) to lock MRAS in its active conformation or employ constitutively active MRAS-Q71L mutant to enhance complex formation .

What are the considerations when studying SHOC2 in the context of RASopathies and cancer research?

When studying SHOC2 in RASopathies (such as Noonan syndrome) and cancer research, several important considerations should be addressed:

For RASopathies:

• SHOC2 mutations (particularly S2G, M173I, and Q269H/H270Y) are associated with Noonan syndrome-like with loose anagen hair (NSLAH)

• When using SHOC2 Antibody, HRP conjugated in patient-derived samples, researchers should verify that the antibody recognizes the mutant forms by including appropriate controls

• The antibody can be used to assess SHOC2 expression and localization changes in patient samples via IHC-P (1:200-400 dilution)

For cancer research:

• SHOC2 acts as a strong synthetic lethal interactor with MEK inhibitors in KRAS cancer cell lines

• Researchers can use the antibody to:

  • Monitor SHOC2 expression levels in various cancer cell lines via Western blot (1:300-5000 dilution)

  • Evaluate SHOC2 localization changes following treatment with MAPK pathway inhibitors via immunofluorescence

  • Assess SHOC2 expression in patient-derived xenograft models via IHC-P (1:200-400 dilution)

A methodological approach would include parallel analysis of SHOC2 expression, SMP complex formation, and MAPK pathway activation (pERK levels) in response to various perturbations (gene knockdown, MAPK inhibitors, etc.).

How can SHOC2 Antibody be used to investigate the structural elements of SHOC2 and their functional significance?

SHOC2 contains several structural elements that are critical for its function, including leucine-rich repeats (LRRs) and an N-terminal intrinsically disordered region containing an RVxF motif that interacts with PP1C . To investigate these structural elements using SHOC2 Antibody, HRP conjugated, researchers can:

• Generate SHOC2 truncation or point mutation constructs targeting specific domains:

  • N-terminal region (residues 58-86)

  • RVxF motif (residues 62-66, particularly V64 and F66)

  • Individual LRRs or groups of LRRs

  • E155 residue that interacts with R188 of PP1CA

• Express these constructs in SHOC2-knockout cell lines

• Use the SHOC2 Antibody, HRP conjugated (1:1000 dilution for WB) to:

  • Confirm expression of the mutant constructs

  • Assess protein stability of different mutants

  • Determine subcellular localization changes

• Combine with co-IP experiments to determine how structural mutations affect:

  • Interaction with PP1C

  • Interaction with MRAS

  • Formation of the ternary SMP complex

This approach can help elucidate the structure-function relationship of SHOC2 and identify critical residues for protein-protein interactions within the SMP complex.

What are common issues when using SHOC2 Antibody, HRP Conjugated and how can they be resolved?

When working with SHOC2 Antibody, HRP Conjugated, researchers may encounter several common issues. Here are methodological approaches to resolve them:

• High background in Western blots:

  • Increase blocking time (2 hours at room temperature or overnight at 4°C)

  • Use a different blocking agent (5% BSA instead of milk)

  • Increase washing time and number of washes

  • Optimize antibody dilution (start with 1:1000 and adjust as needed)

  • Ensure membranes are completely covered during all incubation steps

• Weak or no signal:

  • Check protein loading (increase if necessary)

  • Reduce washing stringency

  • Increase antibody concentration (use 1:300 dilution)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Verify target protein expression in your sample

  • Ensure the detection substrate is fresh and active

• Non-specific bands:

  • Increase antibody dilution (use 1:5000 dilution)

  • Use freshly prepared lysates

  • Add protease inhibitors during sample preparation

  • Perform peptide competition assay to identify specific bands

• Poor reproducibility:

  • Standardize lysate preparation protocol

  • Aliquot antibody to avoid freeze-thaw cycles

  • Maintain consistent incubation times and temperatures

  • Use the same membrane type and blocking conditions between experiments

For IHC applications, additional optimization steps include adjusting antigen retrieval methods, optimizing antibody concentration (1:200-400 for IHC-P) , and testing different detection systems.

How can researchers optimize the use of SHOC2 Antibody, HRP Conjugated for specific experimental conditions?

Optimizing SHOC2 Antibody, HRP Conjugated usage for specific experimental conditions requires systematic adjustment of several parameters:

• For Western blotting optimization:

  • Antibody dilution: Test a range from 1:300 to 1:5000

  • Protein loading: 10-50 μg total protein per lane

  • Blocking conditions: Compare 5% milk vs. 5% BSA

  • Incubation time: 1 hour at room temperature vs. overnight at 4°C

  • Detection method: Compare standard ECL vs. enhanced sensitivity substrates

  Create an optimization matrix and test each combination systematically.

• For IHC-P optimization:

  • Antigen retrieval: Compare citrate buffer (pH 6.0) vs. EDTA buffer (pH 9.0)

  • Antibody dilution: Test dilutions between 1:200-400

  • Incubation time: 1 hour at room temperature vs. overnight at 4°C

  • Detection system: DAB vs. AEC substrate

  • Counterstaining: Hematoxylin timing optimization

• For ELISA optimization:

  • Coating concentration: 1-10 μg/ml of capture antibody

  • Blocking buffer: Compare BSA vs. normal serum

  • Antibody dilution: Test 1:500-1000

  • Substrate incubation time: 5-30 minutes

For each application, a standard curve of antibody dilutions should be generated using positive control samples to determine the optimal signal-to-noise ratio. Additionally, the specificity of staining should be confirmed using SHOC2 knockdown/knockout samples.

How can SHOC2 Antibody be used to investigate the relationship between SHOC2 and therapeutic resistance in cancer?

SHOC2 has been identified as a potentially important factor in therapeutic resistance, particularly in KRAS-mutant cancers treated with MEK inhibitors . To investigate this relationship using SHOC2 Antibody, HRP conjugated, researchers can implement the following methodological approach:

• Establish resistant cell line models:

  • Develop MEK inhibitor-resistant cancer cell lines through long-term exposure to increasing drug concentrations

  • Maintain parallel sensitive parental lines

• Expression analysis:

  • Use SHOC2 Antibody, HRP conjugated (1:1000 dilution for WB) to compare SHOC2 expression levels between sensitive and resistant cell lines

  • Perform subcellular fractionation to determine if SHOC2 localization differs in resistant cells

• Functional studies:

  • Knockdown SHOC2 in resistant cells and assess re-sensitization to MEK inhibitors

  • Overexpress SHOC2 in sensitive cells and determine if this confers resistance

  • Use the antibody to confirm knockdown or overexpression efficiency

• Combinatorial treatment assessment:

  • Treat cells with MEK inhibitors alone or in combination with inhibitors of the SHOC2-MRAS-PP1C complex

  • Monitor changes in SHOC2 expression and localization

  • Assess downstream MAPK pathway activation

• Patient-derived samples:

  • Use IHC-P (1:200-400 dilution) to analyze SHOC2 expression in tumor samples from patients before treatment and after developing resistance

  • Correlate SHOC2 expression with response duration and patient outcomes

This comprehensive approach would provide insights into how SHOC2 contributes to therapeutic resistance and whether targeting SHOC2 could be a valuable strategy to overcome resistance to MAPK pathway inhibitors.

What methodological considerations are important when using SHOC2 Antibody to investigate the SMP complex in different cell types and tissues?

When investigating the SHOC2-MRAS-PP1C (SMP) complex across different cell types and tissues using SHOC2 Antibody, HRP conjugated, several methodological considerations are important:

• Cell type-specific expression levels:

  • SHOC2 expression varies across cell types

  • Adjust antibody dilution based on expression level (1:300 for low expressers, 1:5000 for high expressers)

  • Include positive control lysates from cells known to express SHOC2 abundantly

• Tissue-specific optimization for IHC:

  • Different tissues may require specific antigen retrieval methods

  • Optimize antibody dilution for each tissue type (starting at 1:200-400 for IHC-P)

  • Consider tissue-specific autofluorescence (for immunofluorescence) or endogenous peroxidase activity (for IHC)

• Complex detection strategies:

  • In tissues or cells where the SMP complex components are expressed at different levels:

    • Use proximity ligation assay (PLA) to detect in situ protein-protein interactions

    • Employ sequential immunoprecipitation to enrich for the complete SMP complex

    • Consider crosslinking approaches to stabilize transient interactions

• Activation state considerations:

  • The SMP complex only forms when MRAS is in its active GTP-bound state

  • Include conditions that activate MRAS in different cell types

  • Consider basal activation states that may differ between cell types

• Subcellular localization differences:

  • SHOC2 can localize to both cytoplasm and nucleus

  • Use subcellular fractionation followed by Western blotting to assess compartmentalization

  • For IHC/IF, carefully analyze localization patterns that may vary by cell type

By addressing these considerations, researchers can effectively use SHOC2 Antibody, HRP conjugated to investigate the SMP complex across diverse cellular contexts and tissue environments.

How should researchers analyze and interpret SHOC2 Antibody data in the context of MAPK pathway dysregulation?

When analyzing and interpreting data generated using SHOC2 Antibody, HRP conjugated in the context of MAPK pathway dysregulation, researchers should consider:

• Quantitative analysis of expression levels:

  • Use densitometry to quantify SHOC2 protein levels in Western blots

  • Normalize to appropriate loading controls (β-actin, GAPDH)

  • Compare expression across:

    • Normal vs. disease tissue

    • Parental vs. resistant cell lines

    • Before vs. after treatment with MAPK pathway inhibitors

• Correlation analysis with MAPK pathway activity:

  • Assess the relationship between SHOC2 expression and:

    • ERK1/2 phosphorylation status

    • RAF1 Ser259 dephosphorylation

    • MRAS activation state

  • Calculate Pearson or Spearman correlation coefficients between SHOC2 levels and these markers

• Statistical considerations:

  • Perform at least three independent experiments

  • Use appropriate statistical tests (t-test, ANOVA, etc.)

  • Consider multiple testing correction for large-scale analyses

  • Report p-values and confidence intervals

• Complex formation analysis:

  • Quantify the relative amounts of SHOC2, MRAS, and PP1C in immunoprecipitates

  • Assess how mutations or treatments affect complex stoichiometry

  • Consider competitive binding scenarios with other PP1C-interacting proteins

• Interpretation frameworks:

  • Loss-of-function context: Determine if SHOC2 reduction correlates with decreased MAPK signaling

  • Gain-of-function context: Assess if SHOC2 overexpression enhances MAPK pathway activity

  • Mutation context: Analyze how SHOC2 mutations affect its function in the MAPK pathway

  • Therapeutic context: Evaluate if SHOC2 modulation sensitizes cells to MAPK pathway inhibitors

By following these analytical approaches, researchers can extract meaningful insights from SHOC2 Antibody data in relation to MAPK pathway dysregulation.

What are the best practices for quantifying SHOC2 expression in different experimental contexts?

Best practices for quantifying SHOC2 expression using the HRP-conjugated antibody across different experimental contexts include:

• Western blot quantification:

  • Use a dilution series of recombinant SHOC2 protein to create a standard curve

  • Ensure samples fall within the linear range of detection (consider multiple exposures)

  • Use digital imaging systems rather than film for better quantitation

  • Normalize SHOC2 signal to total protein (Ponceau S or REVERT stain) or housekeeping proteins

  • Include at least three biological replicates per condition

  • Report relative expression as fold-change from control

• ELISA quantification:

  • Optimize antibody dilution (1:500-1000) for maximum sensitivity and specificity

  • Include a standard curve using recombinant SHOC2 protein

  • Perform technical triplicates for each sample

  • Calculate absolute SHOC2 concentration using the standard curve

  • Validate results with spike-in recovery experiments

• IHC quantification:

  • Use digital pathology software for quantitative analysis

  • Score both staining intensity and percentage of positive cells

  • Develop an H-score (0-300) or Quick score (0-7) system

  • Have multiple independent observers score samples blindly

  • Include appropriate positive and negative controls on each slide

• Flow cytometry quantification:

  • Optimize permeabilization conditions for intracellular SHOC2 detection

  • Use median fluorescence intensity (MFI) for quantification

  • Include fluorescence minus one (FMO) controls

  • Calculate the specific staining index relative to negative controls

• Single-cell analysis considerations:

  • Account for cell-to-cell variability in SHOC2 expression

  • Consider using image cytometry to correlate SHOC2 levels with morphological features

  • Analyze subcellular distribution patterns quantitatively

These best practices ensure reliable, reproducible quantification of SHOC2 expression across diverse experimental platforms.