SH3BGRL Antibody

What is SH3BGRL and what cellular functions does it regulate?

SH3BGRL (SH3 Domain Binding Glutamic Acid-Rich Protein Like) is a small adaptor protein consisting of 114 amino acids that is ubiquitously expressed across various tissues. It plays a crucial role in cellular signaling pathways through its SH3 domain, which facilitates binding to proline-rich motifs in target proteins . This interaction is vital for various cellular processes, including cytoskeletal organization, cell proliferation, and apoptosis, making SH3BGRL essential for maintaining cellular homeostasis and responding to external stimuli . The gene encoding SH3BGRL is located on the X chromosome, which can have implications for sex-specific expression patterns and potential disease associations .

What detection methods are available for SH3BGRL protein analysis?

Multiple validated detection methods are available for SH3BGRL protein:

When selecting an antibody, researchers should consider specificity, host species compatibility, clonality, and validated applications for their experimental system.

What species reactivity is available for SH3BGRL antibodies?

Different SH3BGRL antibodies offer varying species reactivity profiles:

Researchers should select antibodies based on their experimental species model and required applications.

How should I optimize western blotting protocols for SH3BGRL detection?

For optimal SH3BGRL detection via western blotting:

• Sample preparation: Use RIPA buffer with protease inhibitors. Mouse colon and uterus tissues serve as positive controls .

• Protein loading: Load 20-50 μg of total protein per lane. SH3BGRL is 114 amino acids, with expected molecular weight of approximately 12-15 kDa.

• Antibody selection and dilution:

  • Mouse monoclonal E-5 antibody: Dilute 1:50-1:400

  • Rabbit polyclonal antibodies: Follow manufacturer's recommended dilutions

• Incubation conditions:

  • Primary antibody: Overnight at 4°C

  • Secondary antibody: 1-2 hours at room temperature

• Detection systems: Compatible with various detection methods including HRP/chemiluminescence systems and near-infrared imaging platforms .

• Controls: Include positive tissue controls and loading controls (GAPDH, β-actin).

What methods can detect SH3BGRL in fixed tissue samples?

For detecting SH3BGRL in fixed tissues:

• Formalin-fixed paraffin-embedded (FFPE) tissues:

  • Recommended dilution: 1:10-1:100

  • Antigen retrieval: Heat-induced epitope retrieval in appropriate buffer

  • Detection system: HRP/DAB or fluorescence-based detection

  • Counterstaining: Hematoxylin for brightfield or DAPI for fluorescence

• Frozen tissue sections:

  • Recommended dilution: 1:50-1:500

  • Fixation: Post-fixation with paraformaldehyde or acetone

  • Signal amplification: Consider tyramide signal amplification for low-abundance detection

• Controls:

  • Positive controls: Include tissues known to express SH3BGRL (e.g., mouse colon tissue)

  • Negative controls: Omit primary antibody or use isotype controls

  • Peptide competition: Use neutralizing peptide to confirm specificity

How is SH3BGRL involved in cancer progression and chemoresistance?

Recent research has revealed SH3BGRL's critical role in cancer:

• Expression in cancer: SH3BGRL is elevated in the majority of breast cancer patients and correlates with relapse and poor prognosis .

• Chemoresistance mechanism: SH3BGRL upregulation enhances breast cancer cell resistance to doxorubicin through autophagy-mediated protection .

• Molecular pathway: SH3BGRL binds to ribosomal subunits to enhance PIK3C3 translation efficiency and stabilize ATG12, both critical autophagy components .

• Therapeutic implications: Inhibition of autophagy or silencing PIK3C3/ATG12 blocks SH3BGRL-driven doxorubicin resistance in vitro and in vivo .

• Clinical correlations: SH3BGRL expression positively correlates with PIK3C3/ATG12 expression and constitutive autophagy in clinical breast cancer samples .

These findings suggest targeting SH3BGRL could overcome chemoresistance in breast cancer treatment protocols.

What methodological approaches can investigate SH3BGRL-autophagy interactions?

To study SH3BGRL's role in autophagy regulation:

• Protein interaction studies:

  • Co-immunoprecipitation using SH3BGRL antibodies to isolate ribosomal subunits, PIK3C3, and ATG12

  • Proximity ligation assay for visualizing interactions in situ

  • FRET/BRET assays for real-time interaction monitoring

• Translation efficiency analysis:

  • Polysome profiling to analyze PIK3C3 mRNA translation

  • Ribosome footprinting to assess translation at nucleotide resolution

  • Reporter assays with PIK3C3 regulatory elements

• Protein stability measurements:

  • Cycloheximide chase assays to determine ATG12 half-life

  • Proteasomal and lysosomal inhibition studies

  • Ubiquitination assays for post-translational modifications

• Autophagy flux assessment:

  • LC3 conversion (LC3-I to LC3-II) by western blotting

  • Autophagic vesicle visualization using fluorescently-tagged LC3

  • Flux inhibition with Bafilomycin A1 or Chloroquine

• In vivo validation:

  • Patient-derived xenograft models with manipulated SH3BGRL expression

  • Correlation studies in clinical samples using validated antibodies

How can SH3BGRL antibodies help identify potential therapeutic targets in cancer?

SH3BGRL antibodies offer several approaches for therapeutic target identification:

• Expression profiling:

  • IHC screening of tumor microarrays to correlate SH3BGRL with clinical outcomes

  • Analysis of expression in treatment-resistant vs. sensitive tumors

  • Identification of cancer types with SH3BGRL overexpression

• Interactome mapping:

  • Immunoprecipitation coupled with mass spectrometry to identify cancer-specific interaction partners

  • Comparison of SH3BGRL interactome between normal and cancer cells

  • Identification of druggable nodes in SH3BGRL-regulated pathways

• Functional studies:

  • Antibody-mediated neutralization in cellular models

  • Epitope mapping to identify critical functional domains

  • Structure-function analysis for rational drug design

• Biomarker development:

  • Development of assays to quantify SH3BGRL as predictive biomarker for autophagy inhibitor response

  • Multiplex IHC to correlate SH3BGRL with autophagy markers (PIK3C3, ATG12)

  • Liquid biopsy approaches for non-invasive monitoring

How can I troubleshoot non-specific binding or weak signal with SH3BGRL antibodies?

For common technical issues with SH3BGRL antibodies:

• Non-specific binding issues:

  • Increase blocking stringency (5% BSA or milk in TBST)

  • Optimize antibody dilution (test range from 1:50-1:500)

  • Add additional washing steps with increased detergent (0.1-0.3% Tween-20)

  • Consider using the neutralizing peptide for competition controls

  • Use antibodies targeting different epitopes to confirm specificity

• Weak signal problems:

  • Decrease antibody dilution (use more concentrated antibody)

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

  • Ensure proper antigen retrieval for fixed samples

  • Use signal enhancement systems (HRP amplification, tyramide signal amplification)

  • Avoid repeated freeze-thaw cycles of antibody

• Sample considerations:

  • Ensure complete protein denaturation for SDS-PAGE

  • Include protease inhibitors during extraction

  • Use fresh tissue samples or properly stored lysates

  • For mouse colon or uterus tissue as positive controls

What validation methods confirm SH3BGRL antibody specificity?

To validate SH3BGRL antibody specificity:

• Genetic validation:

  • Test antibody in SH3BGRL knockout/knockdown models

  • Compare signal in cells with endogenous vs. overexpressed SH3BGRL

  • Use cells from different species to confirm cross-reactivity claims

• Peptide competition:

  • Pre-incubate antibody with SH3BGRL blocking peptide (e.g., sc-377108 P)

  • Observe proportional signal reduction with increasing peptide concentration

• Multi-antibody validation:

  • Compare results using antibodies targeting different epitopes

  • Use both monoclonal (E-5) and polyclonal antibodies for confirmation

  • Compare different applications (WB, IHC, IF) for consistent results

• Orthogonal methods:

  • Correlate protein detection with mRNA expression data

  • Perform immunoprecipitation followed by mass spectrometry

  • Compare results from multiple detection methods

How can SH3BGRL antibodies be used to study cancer chemoresistance mechanisms?

SH3BGRL antibodies enable multiple experimental approaches to study chemoresistance:

• Patient stratification:

  • IHC analysis of patient samples to correlate SH3BGRL levels with treatment response

  • Development of predictive biomarkers for chemotherapy resistance

  • Identification of patients suitable for autophagy inhibitor combination therapy

• Mechanistic studies:

  • Monitor changes in SH3BGRL-autophagy signaling during acquired resistance

  • Track SH3BGRL interaction with ribosomal subunits during doxorubicin treatment

  • Analyze PIK3C3 translation efficiency and ATG12 stability in resistant vs. sensitive cells

• Therapeutic targeting validation:

  • Antibody-mediated inhibition of SH3BGRL function

  • Correlation of autophagy levels with SH3BGRL expression

  • Combination studies with autophagy inhibitors and conventional chemotherapy

• In vivo monitoring:

  • IHC analysis of xenograft models treated with chemotherapy

  • Correlation of treatment response with SH3BGRL and autophagy markers

  • Longitudinal studies to track resistance development

What considerations are important when studying SH3BGRL in different cell and tissue types?

Important considerations for cell and tissue-specific SH3BGRL research:

• Baseline expression patterns:

  • SH3BGRL is ubiquitously expressed but at varying levels across tissues

  • Mouse colon and uterus tissues show robust expression

  • Consider tissue-specific expression when interpreting cancer-associated changes

• Antibody selection by application:

  • For human samples: Multiple antibody options available

  • For mouse/rat studies: Select antibodies with validated cross-reactivity

  • For cross-species studies: Consider antibodies targeting conserved epitopes

• Context-dependent function:

  • SH3BGRL may have tissue-specific interacting partners

  • Autophagy regulation may vary across cell types

  • Cancer-specific alterations may differ from normal function

• Experimental controls:

  • Include tissue-matched controls when studying cancer samples

  • Use appropriate cell line models that reflect tissue of origin

  • Consider genetic background when using engineered cell lines

How can SH3BGRL expression be quantified in clinical samples for biomarker development?

For clinical biomarker development using SH3BGRL:

• Immunohistochemistry approaches:

  • H-score method (intensity × percentage of positive cells)

  • Digital pathology with automated scoring systems

  • Multiplex IHC to correlate with autophagy markers (PIK3C3, ATG12)

  • Standardization across laboratories with validated protocols

• Quantitative protein analysis:

  • ELISA-based quantification (1:100-1:5000 antibody dilution)

  • Western blotting with densitometry and standard curves

  • Protein array technologies for high-throughput analysis

  • Mass spectrometry for absolute quantification

• mRNA expression correlation:

  • qRT-PCR for SH3BGRL transcript levels

  • RNA-seq for comprehensive expression profiling

  • Correlation of protein and mRNA levels to identify post-transcriptional regulation

• Standardization considerations:

  • Use of calibration standards across experiments

  • Inclusion of reference samples in each batch

  • Development of clinical-grade assays with defined cut-off values

  • Validation in multi-center studies