SMG7 Antibody, HRP conjugated

What experimental applications are suitable for HRP-conjugated SMG7 antibodies?

HRP-conjugated SMG7 antibodies are versatile tools applicable to numerous experimental techniques:

• ELISA: The primary application listed for commercial HRP-conjugated SMG7 antibodies, allowing quantitative measurement of SMG7 protein levels .

• Western blotting: Enables detection of SMG7 protein expression and post-translational modifications without requiring secondary antibody incubation.

• Immunocytochemistry/Immunohistochemistry: For visualization of SMG7 cellular localization and expression patterns.

• Protein interaction studies: Can be used in pull-down assays to investigate SMG7's interactions with binding partners like RAD17, UPF1, and SMG5 .

• Chromatin association studies: SMG7 constitutively associates with chromatin, making these antibodies useful for examining SMG7's recruitment to DNA damage sites .

When designing experiments, researchers should consider that antibodies targeting specific epitopes (like AA 694-809) may have limitations in certain applications if the epitope becomes masked in protein complexes.

How can I validate the specificity of an SMG7 antibody in my experimental system?

Thorough validation of SMG7 antibody specificity requires a multi-faceted approach:

• Genetic validation: Compare antibody signal between wild-type cells and SMG7 knockout models (such as HCT116 SMG7-/- cells mentioned in the literature) .

• siRNA knockdown: Perform siRNA-mediated SMG7 knockdown experiments as demonstrated in the literature, where Accell siRNAs targeting SMG7 (E-021305) were used to validate specificity .

• Competing peptide blocking: Pre-incubate the antibody with excess recombinant SMG7 peptide (694-809AA) corresponding to the immunogen sequence .

• Western blot analysis: Verify single band of appropriate molecular weight (~130 kDa for full-length SMG7).

• Cross-reactivity assessment: Test antibody reactivity in human samples as well as samples from other species if cross-reactivity is claimed.

• Positive controls: Include samples known to express high levels of SMG7, such as cell lines used in published studies (HCT116, H1299).

• Application-specific validation: For each application (ELISA, western blot, etc.), perform specific controls appropriate for that technique.

Documentation of these validation steps is essential for result reproducibility and reliability in SMG7 research.

What is the significance of the AA 694-809 region of SMG7 targeted by this antibody?

The antibody targeting the AA 694-809 region of SMG7 recognizes a functionally relevant portion of the protein that contains important structural and regulatory elements:

• This region falls within the C-terminal low complexity region (LCR) of SMG7, which has been shown to interact with certain binding partners .

• While not containing the N-terminal 14-3-3 domain that mediates interactions with phosphorylated proteins like RAD17, p53, and UPF1, this region may participate in protein-protein interactions that regulate SMG7 function .

• The C-terminal region of SMG7 is involved in its localization and stability, potentially affecting its role in nonsense-mediated mRNA decay and other cellular processes.

• Antibodies targeting this region are less likely to be affected by N-terminal post-translational modifications that might mask epitopes in the 14-3-3 domain during phosphorylation-dependent signaling events.

• This region is sufficiently unique to SMG7 to minimize cross-reactivity with related proteins like SMG5, enhancing specificity in experimental applications.

Understanding the structural context of this epitope is crucial when interpreting experimental results, particularly when studying SMG7's role in multi-protein complexes where this region might be involved in interactions.

What advantages does HRP conjugation provide for SMG7 antibody applications?

HRP (horseradish peroxidase) conjugation offers several significant advantages for SMG7 antibody applications:

• Increased sensitivity: HRP enzymatic amplification enables detection of low-abundance SMG7 protein, particularly important when studying cells with decreased SMG7 expression as observed in certain disease states .

• Streamlined workflows: Direct conjugation eliminates the need for secondary antibody incubation steps, reducing experimental time and potential background.

• Quantitative applications: HRP-conjugated antibodies are particularly well-suited for quantitative ELISA development to measure SMG7 protein levels across different experimental conditions .

• Multiplexing capability: When studying SMG7 alongside other proteins (like its binding partners RAD17, UPF1, etc.), HRP-conjugated antibodies can be paired with antibodies using different detection systems.

• Chemiluminescent or colorimetric detection: Compatible with various substrate systems, providing flexibility in detection methods depending on available equipment.

• Enhanced signal-to-noise ratio: Direct conjugation often results in cleaner backgrounds compared to multi-step detection systems, particularly valuable when studying chromatin-associated fractions of SMG7.

When using HRP-conjugated antibodies, researchers should implement measures to prevent signal loss due to potential HRP denaturation during storage and experimental procedures.

What controls should I include when designing SMG7 antibody experiments?

Robust experimental design for SMG7 antibody-based studies requires several critical controls:

• Negative controls:

  • Isotype control antibody (rabbit polyclonal IgG with HRP conjugation)

  • Non-targeting siRNA controls when performing SMG7 knockdown experiments (D-001910 has been validated)

  • SMG7 knockout cells (HCT116 SMG7-/- are described in literature)

  • Secondary antibody-only controls (for non-conjugated applications)

• Positive controls:

  • Cell lines with confirmed SMG7 expression (H1299 cells expressing FH-SMG7)

  • Recombinant SMG7 protein (particularly fragments containing AA 694-809)

  • Cells treated with agents known to activate pathways involving SMG7 (ionizing radiation for DNA damage response)

• Specificity controls:

  • Peptide competition with immunogen (SMG7 protein 694-809AA)

  • Parallel detection with alternative SMG7 antibodies targeting different epitopes

  • Gradient of SMG7 expression (via siRNA knockdown or overexpression)

• Technical controls:

  • Loading controls for western blotting (GAPDH, β-actin)

  • Inter-assay calibration samples for quantitative applications

  • Medium-only mock controls for cell-based assays

Documenting these controls thoroughly ensures the reliability and reproducibility of SMG7-related findings.

How should SMG7 HRP-conjugated antibodies be stored and handled to maintain optimal activity?

Proper storage and handling of SMG7 HRP-conjugated antibodies are essential for maintaining their activity and ensuring experimental consistency:

• Storage conditions:

  • Store at 2-8°C for short-term (1-2 weeks) preservation

  • For long-term storage, aliquot and maintain at -20°C to prevent freeze-thaw cycles

  • Avoid storing diluted antibody solutions as protein concentration affects stability

• Buffer considerations:

  • Store in buffers containing stabilizing proteins (BSA, gelatin)

  • Avoid repeated exposure to strong acids or bases that may denature the antibody

  • Consider adding preservatives (0.02% sodium azide or thimerosal) for solutions stored at 4°C

• HRP-specific precautions:

  • Minimize exposure to oxidizing agents that can inactivate the HRP enzyme

  • Protect from prolonged light exposure when in solution

  • Avoid contamination with transition metals that may catalyze peroxidase reactions

• Working practices:

  • Centrifuge briefly before opening vials to collect liquid at the bottom

  • Use only clean pipette tips dedicated to antibody handling

  • Return to recommended storage immediately after use

  • Document date of first use and number of freeze-thaw cycles

• Stability assessment:

  • Periodically verify activity using positive control samples

  • Monitor for changes in background signal that might indicate deterioration

  • Consider preparing new working aliquots if signal quality decreases

Following these guidelines will help maximize antibody performance and extend the useful life of SMG7 HRP-conjugated antibodies in research applications.

How can I use SMG7 antibodies to investigate nonsense-mediated mRNA decay pathways?

Investigating SMG7's role in nonsense-mediated mRNA decay (NMD) requires a comprehensive experimental approach:

• Protein complex analysis:

  • Use SMG7 antibodies for co-immunoprecipitation to isolate protein complexes containing known NMD factors like UPF1 and SMG5

  • Combine with mass spectrometry to identify novel interaction partners

  • Perform proximity ligation assays to visualize SMG7-UPF1 interactions in situ

• Target transcript identification:

  • Couple SMG7 knockdown with RNA-seq to identify transcripts regulated by SMG7-dependent NMD

  • Validate findings using RT-qPCR for specific transcripts of interest

  • Compare profiles between wild-type and SMG7-/- cells, focusing on both PTC-containing mRNAs and lncRNAs that showed preferential overexpression in SMG7-deficient cells

• Functional domain studies:

  • Utilize SMG7 antibodies in combination with domain-specific mutations to determine regions required for NMD activity

  • Investigate how the 14-3-3 domain interactions affect NMD efficiency

  • Examine if phosphorylation status of SMG7 binding partners impacts NMD function

• Pathway dynamics:

  • Use SMG7 antibodies to track protein localization during active NMD

  • Implement pulse-chase experiments to examine the kinetics of SMG7 recruitment to NMD complexes

  • Analyze how stress conditions affect SMG7's role in NMD using various cellular stressors

• Disease-relevant NMD regulation:

  • Investigate how SMG7 levels correlate with NMD efficiency in disease models

  • Examine the relationship between SMG7 expression and immune responses in autoimmune conditions

  • Analyze SMG7-dependent regulation of oncogenic transcripts in cancer cell models

This multifaceted approach leverages SMG7 antibodies to dissect the complex regulatory mechanisms of NMD pathways in normal and disease states.

What technical considerations are important when studying SMG7's interaction with RAD17 in DNA damage response?

Investigating the SMG7-RAD17 interaction in DNA damage response requires careful experimental design:

• Phosphorylation-dependent binding analysis:

  • SMG7's 14-3-3 domain interacts specifically with RAD17 phosphorylated at S635 by ATR kinase

  • Include phosphatase treatments as negative controls to demonstrate phosphorylation-dependency

  • Utilize phospho-specific RAD17 (S635) antibodies alongside SMG7 antibodies in co-immunoprecipitation studies

  • Compare binding between wild-type RAD17 and S635A mutants that cannot be phosphorylated

• Kinetic analysis of complex formation:

  • Track temporal dynamics of SMG7-RAD17 interaction following DNA damage induction

  • Utilize time-course experiments with various genotoxic agents (ionizing radiation, UV, hydroxyurea)

  • Correlate complex formation with ATR activation kinetics

  • Compare protein complex stability in different cell cycle phases

• Structural considerations:

  • The N-terminal 14-3-3 domain of SMG7 directly binds RAD17

  • AA 694-809 targeted by some antibodies lies outside this interaction domain

  • Select appropriate antibodies that do not interfere with the binding interface

  • Consider epitope accessibility in chromatin-bound complexes

• Functional consequence assessment:

  • Evaluate RAD9 recruitment to DNA damage sites in presence/absence of SMG7

  • Measure CHK1 phosphorylation as downstream readout of pathway activation

  • Utilize domain-specific SMG7 mutants to dissect required regions for RAD17 interaction

  • Monitor γ-H2AX dynamics as marker of DNA damage resolution

• Experimental system selection:

  • HCT116 SMG7-/- cells have been validated for studying DNA damage response

  • H1299 cells have been used successfully for SMG7-RAD17 interaction studies

  • Consider cell type-specific differences in ATR-CHK1 pathway regulation

Implementing these considerations will help generate reliable data on the SMG7-RAD17 interaction in DNA damage response signaling.

How can SMG7 antibodies be utilized to explore TNFα-induced apoptosis pathways?

Investigating SMG7's role in TNFα-induced apoptosis requires a strategic experimental approach:

• Pathway component analysis:

  • Use SMG7 antibodies to track changes in protein levels/localization during TNFα treatment

  • Examine relationships between SMG7 and NF-κB activation through co-immunoprecipitation of pathway components

  • Investigate interactions between SMG7 and the tumor suppressor CYLD, which has shown a positive correlation with SMG7 expression in multiple cancer cell lines

  • Monitor changes in SMG7-regulated lncRNAs (Pvt1 and Adapt33) that confer resistance to TNFα-induced cell death

• Functional consequence assessment:

  • Compare TNFα responses between wild-type and SMG7-deficient cells

  • Measure caspase activity as a downstream apoptotic readout, which is diminished in Smg7-/- cells

  • Assess NF-κB activation through nuclear translocation, target gene expression, and reporter assays

  • Utilize 3D spheroid models to evaluate cell survival and compaction phenotypes observed in SMG7-deficient conditions

• Regulatory circuit manipulation:

  • Combine SMG7 antibody-based detection with overexpression/knockdown of key components:

    • CYLD restoration in SMG7-deficient cells

    • Manipulation of Pvt1 and Adapt33 lncRNA levels

    • Pharmacological inhibition of NF-κB to re-establish TNFα sensitivity

  • Track protein complex formation during pathway manipulation

• Translational relevance assessment:

  • Examine correlation between SMG7 and CYLD expression in patient-derived samples

  • Analyze survival outcomes in relation to SMG7 expression levels

  • Use tissue microarrays with SMG7 antibodies to assess expression patterns across tumor types

• Mechanistic dissection:

  • Determine whether SMG7's role in TNFα response depends on its NMD function

  • Investigate if 14-3-3 domain interactions are required for regulating TNFα-induced apoptosis

  • Explore potential cross-talk between DNA damage response and TNFα signaling pathways

This comprehensive approach will help elucidate the complex role of SMG7 in regulating the balance between apoptosis and NF-κB-mediated survival in response to TNFα.

What methodological approaches are recommended for studying SMG7's role in autoimmune conditions?

Investigating SMG7's involvement in autoimmune diseases requires specialized experimental approaches:

• Expression correlation studies:

  • Utilize SMG7 antibodies to quantify protein levels in patient-derived PBMCs compared to healthy controls

  • Correlate SMG7 expression with disease biomarkers like ANA titers, which have shown inverse correlation with SMG7 levels in SLE patients (r=-0.31, P=0.01)

  • Analyze SMG7 expression in relation to specific SLE-associated SNPs, particularly rs2275675 in the SMG7 promoter region that associates with decreased SMG7 mRNA levels

• Functional knockdown experiments:

  • Implement siRNA-mediated SMG7 knockdown in PBMCs as demonstrated in published protocols (using Accell siRNAs targeting SMG7, E-021305)

  • Measure multiple immune parameters following knockdown:

    • Autoantibody production (ANA IgG levels increased by 8.0% in SMG7 siRNA-treated cells)

    • Chemokine production (CCL19 levels increased by 150% following SMG7 knockdown)

    • Additional cytokines (CXCL10, IL-6, IL-17, BAFF, IFN-α)

  • Include appropriate controls (GAPDH siRNA as positive control, non-targeting sequences as negative control)

• Patient stratification approaches:

  • Select patients with appropriate clinical characteristics:

    • Seropositive for ANA

    • Inactive disease (SLE Disease Activity Index score <4)

    • Low medication doses (prednisone <15 mg/day) to minimize treatment effects

  • Compare SMG7 levels across patient subgroups with varying disease manifestations

• Genetic association validation:

  • Utilize genotyping data to correlate SNP variants with SMG7 expression levels

  • Perform allele-specific expression analysis for SLE-associated SNPs

  • Implement luciferase reporter assays to validate functional effects of promoter variants

• Mechanistic investigation:

  • Explore the relationship between NMD efficiency and autoantibody production

  • Investigate how decreased SMG7 affects accumulation of mRNA ribonucleoprotein particles (mRNPs) that might serve as autoantigens

  • Examine potential roles in regulating inflammatory gene expression

These methodological approaches will help elucidate the complex role of SMG7 in autoimmune pathogenesis, particularly in SLE.

How can SMG7 antibodies be applied to study the relationship between SMG7 and lncRNA regulation?

Investigating SMG7's role in lncRNA regulation requires specialized experimental strategies:

• Differential expression analysis:

  • Compare lncRNA expression profiles between wild-type and SMG7-deficient cells

  • Focus on specific lncRNAs identified in previous studies, particularly Pvt1 and Adapt33, which showed robust protection against TNFα when overexpressed

  • Utilize RNA-seq coupled with SMG7 knockdown/knockout to identify the broader spectrum of SMG7-regulated lncRNAs

• Mechanistic investigation:

  • Perform RNA immunoprecipitation (RIP) using SMG7 antibodies to identify directly bound lncRNA targets

  • Implement CLIP-seq (Cross-Linking Immunoprecipitation) to map SMG7 binding sites on lncRNAs at nucleotide resolution

  • Determine if SMG7 regulation of lncRNAs depends on the canonical NMD pathway or represents a distinct function

• Structure-function analysis:

  • Generate domain-specific SMG7 mutants to identify regions required for lncRNA regulation

  • Investigate whether the 14-3-3 domain mediates interactions with RNA-binding proteins involved in lncRNA metabolism

  • Examine if C-terminal regions (including AA 694-809) participate in lncRNA-related functions

• Functional consequence assessment:

  • Evaluate phenotypic outcomes of manipulating SMG7-regulated lncRNAs:

    • Cell survival in response to TNFα

    • NF-κB pathway activation

    • Impact on CYLD expression and function

  • Utilize 3D spheroid models to assess more physiologically relevant responses

• Translational relevance:

  • Analyze correlation between SMG7 expression and lncRNA levels (PVT1 in particular) in patient samples

  • Investigate the prognostic significance of SMG7-lncRNA regulatory relationships in cancer

  • Examine potential therapeutic implications of targeting this regulatory axis

This comprehensive approach will help elucidate the complex interplay between SMG7 and lncRNA regulation in both normal and disease states.

What are optimal experimental designs for studying SMG7's potential tumor suppressor role?

Investigating SMG7's potential tumor suppressor function requires systematic experimental approaches:

• Expression correlation analysis:

  • Utilize SMG7 antibodies to assess protein expression across cancer cell lines and patient samples

  • Correlate SMG7 expression with established tumor suppressors like CYLD, which has shown a positive correlation with SMG7 in human cancer cell lines and renal carcinoma samples

  • Analyze publicly available cancer genomics databases (TCGA) for SMG7 alterations and their association with patient outcomes

• Pathway integration studies:

  • Investigate SMG7's regulation of multiple oncogenic pathways:

    • TNFα-induced apoptosis resistance mechanisms

    • NF-κB pathway activation through CYLD downregulation

    • Oncogenic lncRNA (PVT1) expression control

    • p53 pathway regulation through ATM-mediated signaling

  • Use pharmacological pathway inhibitors to define dependencies (NF-κB inhibitors restored TNFα sensitivity in SMG7-deficient cells)

• In vitro transformation assays:

  • Compare colony formation, soft agar growth, and migration/invasion between wild-type and SMG7-deficient cells

  • Investigate cell cycle regulation and genomic stability in SMG7-altered backgrounds

  • Assess resistance to various apoptotic stimuli beyond TNFα

  • Implement 3D spheroid models which have revealed distinct survival phenotypes in SMG7-deficient conditions

• In vivo modeling:

  • Develop xenograft studies comparing tumor growth rates of wild-type versus SMG7-depleted cells

  • Analyze tumor histology and molecular markers (proliferation, apoptosis, NF-κB activity)

  • Consider genetic mouse models with tissue-specific SMG7 alteration

  • Evaluate response to therapies targeting pathways regulated by SMG7

• Mechanistic dissection:

  • Determine whether tumor suppressor functions depend on:

    • NMD activity

    • Specific protein-protein interactions through the 14-3-3 domain

    • DNA damage response signaling via RAD17-ATR-CHK1 axis

    • Regulation of oncogenic lncRNAs like PVT1

This comprehensive approach will help establish the mechanisms and contexts in which SMG7 functions as a tumor suppressor, potentially leading to novel therapeutic strategies targeting these pathways.

How can I optimize detection of SMG7-protein complexes in chromatin fractions?

Optimizing detection of chromatin-associated SMG7 complexes requires specialized approaches:

• Chromatin fractionation optimization:

  • SMG7 constitutively associates with chromatin , requiring careful extraction protocols

  • Implement stepwise extraction methods to separate soluble nuclear proteins from chromatin-bound fractions

  • Consider using nuclease treatments (DNase, benzonase) to release DNA-bound protein complexes

  • Compare different detergent concentrations to optimize extraction while preserving protein-protein interactions

• Crosslinking strategies:

  • Utilize formaldehyde cross-linking to capture transient protein-DNA and protein-protein interactions

  • Consider dual crosslinking approaches (DSP or EGS followed by formaldehyde) for enhanced complex stability

  • Optimize crosslinking time and concentration to prevent over-crosslinking while maintaining complex integrity

  • Include appropriate reversal steps to ensure antibody accessibility to epitopes

• Immunoprecipitation enhancements:

  • Utilize tandem affinity purification approaches as demonstrated with Flag-HA tagged SMG7

  • Implement two-step IP protocols targeting different complex components (e.g., SMG7 followed by RAD17)

  • Consider native versus denaturing conditions depending on the strength of protein interactions

  • Optimize buffer compositions to maintain chromatin-associated complex integrity

• Detection system optimization:

  • HRP-conjugated antibodies can enhance sensitivity for low-abundance chromatin-bound complexes

  • Utilize enhanced chemiluminescence or fluorescent secondary antibodies for maximum detection sensitivity

  • Consider proximity ligation assays for in situ visualization of protein-protein interactions on chromatin

  • Implement mass spectrometry for unbiased identification of SMG7-associated proteins in chromatin fractions

• Specialized applications:

  • Chromatin immunoprecipitation (ChIP) to identify DNA regions associated with SMG7 complexes

  • ChIP-seq for genome-wide mapping of SMG7 chromatin associations

  • Re-ChIP (sequential ChIP) to identify genomic regions with co-occupancy of SMG7 and binding partners

  • FAIRE-seq (Formaldehyde-Assisted Isolation of Regulatory Elements) to correlate SMG7 binding with chromatin accessibility

These optimized approaches will facilitate detection of SMG7's chromatin-associated interactions, particularly with DNA damage response proteins like RAD17 and the 9-1-1 complex.

What techniques can help resolve contradictory findings about SMG7 function across different cell types?

Resolving contradictory findings about SMG7 function requires systematic comparative approaches:

• Standardized experimental systems:

  • Implement parallel studies in multiple cell types using identical methodologies

  • Include validated model systems from published studies:

    • HCT116 and SMG7-/- HCT116 cells for DNA damage response studies

    • H1299 cells for SMG7-RAD17 interaction analysis

    • PBMCs for autoimmune-related functions

  • Develop isogenic cell line panels with precisely controlled SMG7 expression levels

• Context-dependent activation analysis:

  • Systematically vary experimental conditions to identify contextual requirements:

    • Cell cycle phase (synchronize cells to compare SMG7 function across G1, S, G2/M)

    • Growth conditions (serum levels, confluence, 2D vs. 3D culture)

    • Stress states (genotoxic stress, inflammatory stimuli, metabolic stress)

  • Quantitatively compare SMG7 complex formation across these contexts

• Isoform and post-translational modification profiling:

  • Determine if cell type-specific SMG7 isoforms exist using isoform-specific antibodies

  • Map phosphorylation and other modifications across cell types using mass spectrometry

  • Correlate modifications with functional outcomes in different cellular contexts

  • Generate modification-specific antibodies for critical regulatory sites

• Interaction network mapping:

  • Perform comprehensive interactome analysis across cell types:

    • Immunoprecipitation coupled with mass spectrometry

    • Proximity labeling approaches (BioID, APEX)

    • Yeast two-hybrid screening with cell type-specific cDNA libraries

  • Construct interaction networks to identify cell type-specific partners

• Functional redundancy assessment:

  • Investigate compensatory mechanisms through:

    • Combined knockdown of SMG7 with related proteins (SMG5, SMG6)

    • Overexpression of potential redundant factors in SMG7-deficient backgrounds

    • Cross-rescue experiments between cell types showing different phenotypes

• Integrative multi-omics approaches:

  • Combine transcriptomics, proteomics, and phosphoproteomics data across cell types

  • Implement systems biology approaches to model context-dependent SMG7 functions

  • Identify cell type-specific regulatory circuits that modify SMG7 activity

This systematic approach will help resolve contradictory findings and establish a unified model of SMG7 function that accounts for cell type-specific contexts and regulatory mechanisms.