SDCBP2 Antibody

What is SDCBP2 and what cellular functions does it perform?

SDCBP2, also known as Syntenin-2, is a protein encoded by the SDCBP2 gene located on chromosome 20p13 . It contains two class II PDZ domains that facilitate protein-protein interactions by binding to the cytoplasmic C-terminus of transmembrane proteins . SDCBP2 binds to phosphatidylinositol 4,5-bisphosphate (PIP2) and plays a significant role in nuclear PIP2 organization and cell division . The protein has a calculated molecular weight of approximately 32-37 kDa .

SDCBP2 is involved in multiple cellular processes including:

• Cell signaling and organization of protein complexes

• Intracellular transport

• Nervous system development

• Protein homodimerization and heterodimerization activities

Alternative spliced transcript variants encoding multiple isoforms have been observed for the SDCBP2 gene, and read-through transcription exists between this gene and the upstream FKBP1A gene .

What applications are SDCBP2 antibodies most commonly used for?

SDCBP2 antibodies have been validated for multiple research applications, with varying protocols and optimizations for each technique:

For optimal results, validation of antibody specificity and optimization of protocols for each specific experimental system is strongly recommended .

How should I optimize Western blot protocols for SDCBP2 detection?

For successful Western blot detection of SDCBP2, follow these methodological considerations:

• Sample preparation: Use 30 μg of protein lysate per lane under reducing conditions .

• Gel electrophoresis: Run samples on a 5-20% SDS-PAGE gel at 70V (stacking gel) followed by 90V (resolving gel) for 2-3 hours .

• Transfer conditions: Transfer proteins to nitrocellulose membrane at 150 mA for 50-90 minutes .

• Blocking: Block the membrane with 5% non-fat milk in TBS for 1.5 hours at room temperature .

• Primary antibody incubation: Incubate with anti-SDCBP2 antibody at 0.5-1 μg/ml overnight at 4°C .

• Washing: Wash with TBS-0.1% Tween 3 times, 5 minutes each .

• Secondary antibody incubation: Use appropriate HRP-conjugated secondary antibody (e.g., anti-rabbit IgG) at 1:5000 dilution for 1.5 hours at room temperature .

• Detection: Develop using enhanced chemiluminescence (ECL) detection system .

Validation data shows SDCBP2 detection at approximately 37 kDa in human A431 and HaCaT whole cell lysates, though the expected band size is 32 kDa .

What tissue expression patterns have been reported for SDCBP2?

Immunohistochemical analyses have revealed SDCBP2 expression in multiple human tissues and cancer types:

Expression levels may vary based on tissue type, disease state, and individual patient characteristics. Clinical data from 132 gastric cancer patients showed that SDCBP2-AS1 expression correlates with clinical parameters including gender and tumor diameter .

What is the role of SDCBP2 in cancer biology and potential therapeutic targeting?

SDCBP2 and its antisense RNA (SDCBP2-AS1) have emerging roles in cancer biology, particularly in gastric cancer:

• Gastric cancer mechanisms: SDCBP2-AS1 is involved in gastric cancer tumorigenesis and progression through post-translational modifications . Research shows that SDCBP2-AS1 destabilizes β-catenin by regulating ubiquitination processes .

• Clinical correlations: In a study of 132 pairs of gastric cancer and adjacent normal tissues, SDCBP2-AS1 expression showed significant correlation with gender (p=0.019) and tumor diameter (p=0.016) . The following data demonstrates these clinical correlations:

  Characteristic	Total [cases (%)]	Low Expression [cases (%)]	High Expression [cases (%)]	P-value
  Total	132	73 (55.3)	59 (44.7)	-
  Gender	-	-	-	0.019
  Male	88 (66.7)	55 (62.5)	33 (37.5)	-
  Female	44 (33.3)	18 (40.9)	26 (59.1)	-
  Tumor diameter	-	-	-	0.016
  ≤4 cm	63 (47.7)	28 (44.4)	35 (55.6)	-

• Experimental approaches: Investigating SDCBP2's role in cancer requires multiple methodologies:

  • RNA pull-down and immunoprecipitation assays to clarify interactions with other proteins

  • RNA-sequencing to investigate downstream effects

  • Luciferase analysis for promoter activity

  • In vitro and in vivo functional assessments

Understanding these mechanisms provides potential for therapeutic targeting and biomarker development in gastric and other cancers.

How can I design experiments to investigate SDCBP2 protein-protein interactions?

To effectively study SDCBP2 protein-protein interactions, consider the following methodological approach:

• Co-immunoprecipitation (Co-IP):

  • Use anti-SDCBP2 antibodies to pull down protein complexes from cell lysates

  • Perform reciprocal IP with antibodies against suspected interaction partners

  • Analyze precipitated complexes by Western blot or mass spectrometry

• Proximity ligation assay (PLA):

  • Utilize two primary antibodies from different species targeting SDCBP2 and potential binding partners

  • Apply species-specific PLA probes and perform rolling circle amplification

  • Visualize interaction signals using fluorescence microscopy

• PDZ domain interaction mapping:

  • Design experiments focusing on the two class II PDZ domains of SDCBP2

  • Create domain-specific mutations to identify critical interaction regions

  • Use peptide arrays with C-terminal sequences of potential transmembrane protein partners

• Analyzing PIP2 interactions:

  • Employ lipid binding assays to study SDCBP2-PIP2 interactions

  • Use fluorescently labeled PIP2 and purified SDCBP2 protein

  • Perform subcellular fractionation to assess nuclear versus cytoplasmic interactions

When conducting these experiments, it's critical to incorporate appropriate controls and validate results through multiple complementary techniques to ensure specificity of detected interactions.

What methodological approaches are recommended for studying SDCBP2's role in ubiquitination pathways?

Based on research showing SDCBP2-AS1's involvement in regulating ubiquitination , the following experimental approaches are recommended:

• Ubiquitination assays:

  • Perform in vitro ubiquitination reactions using purified components

  • Use co-transfection of SDCBP2, substrate proteins, and tagged ubiquitin

  • Analyze by immunoprecipitation followed by Western blotting with anti-ubiquitin antibodies

• Proteasome inhibition studies:

  • Treat cells with proteasome inhibitors (MG132, bortezomib) to accumulate ubiquitinated proteins

  • Compare SDCBP2 knockdown/overexpression effects on substrate stability

  • Monitor half-life changes of target proteins using cycloheximide chase assays

• E3 ligase interaction analysis:

  • Investigate SDCBP2 interactions with E3 ubiquitin ligases

  • Map binding domains using truncation mutants

  • Perform functional assays to determine if SDCBP2 modulates E3 ligase activity

• Mass spectrometry approaches:

  • Use SILAC or TMT labeling to quantify changes in the ubiquitinome upon SDCBP2 manipulation

  • Enrich for ubiquitinated peptides using K-ε-GG antibodies

  • Identify specific lysine residues modified in a SDCBP2-dependent manner

When interpreting results, consider that SDCBP2 may function as a scaffold that brings together substrate proteins and ubiquitination machinery rather than possessing intrinsic enzymatic activity.

How can I distinguish between specific and non-specific binding when using SDCBP2 antibodies?

Distinguishing specific from non-specific binding is crucial for generating reliable data with SDCBP2 antibodies. Implement these methodological controls and validation steps:

• Knockout/knockdown validation:

  • Compare antibody signals in SDCBP2 knockout/knockdown cells versus wild-type cells

  • Use CRISPR-Cas9, siRNA, or shRNA approaches to deplete SDCBP2

  • Expect significant reduction or elimination of specific signals

• Peptide competition assays:

  • Pre-incubate antibody with excess immunizing peptide before application

  • Compare signals with and without peptide competition

  • Specific signals should be blocked by the peptide

• Multiple antibody validation:

  • Use antibodies raised against different epitopes of SDCBP2

  • Compare staining patterns and detection profiles

  • Consistent results across antibodies suggest specific detection

• Positive and negative control samples:

  • Include tissues/cell lines known to express or lack SDCBP2

  • For Western blots, recombinant SDCBP2 protein can serve as a positive control

  • Antibody validation data shows SDCBP2 expression in multiple cancer cell lines including A431 and HaCaT

• Isotype controls:

  • For flow cytometry and IHC applications, use matched isotype controls

  • Process in parallel with the same concentration as the SDCBP2 antibody

  • This controls for non-specific binding of antibody isotype

By implementing these validation approaches, researchers can confidently distinguish between specific SDCBP2 signals and non-specific background.

What are the optimal protocols for detecting SDCBP2 in fixed tissue sections?

For optimal immunohistochemical detection of SDCBP2 in paraffin-embedded tissue sections, follow this detailed protocol:

• Sample preparation:

  • Use formalin-fixed, paraffin-embedded tissue sections (4-6 μm thickness)

  • Mount on positively charged slides

  • Deparaffinize and rehydrate through xylene and graded ethanol series

• Antigen retrieval:

  • Heat-mediated antigen retrieval in EDTA buffer (pH 8.0)

  • Use pressure cooker or microwave methods

  • Allow sections to cool slowly to room temperature

• Blocking:

  • Block with 10% goat serum for 30-60 minutes at room temperature

  • This reduces non-specific binding of secondary antibodies

• Primary antibody incubation:

  • Apply anti-SDCBP2 antibody at 2-5 μg/ml

  • Incubate overnight at 4°C in a humidified chamber

  • For co-localization studies, combine with other compatible primary antibodies

• Secondary antibody and detection:

  • Use appropriate HRP-conjugated secondary antibody (anti-rabbit/goat IgG)

  • Incubate for 30 minutes at 37°C

  • Develop using DAB as the chromogen

  • Counterstain with hematoxylin

  • Mount with appropriate mounting medium

This protocol has been validated with SDCBP2 antibodies in multiple cancer tissues including breast, colon, liver, lung, prostate, and renal cancers, as well as normal tissues like spleen .

How should I design experiments to investigate SDCBP2 subcellular localization?

To effectively investigate SDCBP2 subcellular localization, implement these methodological approaches:

• Immunofluorescence microscopy:

  • Use anti-SDCBP2 antibodies (5 μg/ml) for detection

  • Co-stain with established subcellular markers:

    • Nuclear: DAPI or anti-lamin antibodies

    • Cytoplasmic: anti-tubulin antibodies

    • Membrane: wheat germ agglutinin or membrane-specific markers

  • Analyze using confocal microscopy for precise localization

• Subcellular fractionation:

  • Separate cellular components (nuclear, cytoplasmic, membrane fractions)

  • Analyze SDCBP2 distribution by Western blotting

  • Include fraction-specific markers as controls (e.g., lamin for nuclear fraction, GAPDH for cytoplasmic fraction)

• Live-cell imaging:

  • Generate cells expressing SDCBP2-GFP/RFP fusion proteins

  • Monitor dynamic localization changes in response to stimuli

  • Combine with photobleaching techniques (FRAP/FLIP) to assess protein mobility

• Super-resolution microscopy:

  • Apply techniques like STORM, PALM, or STED for nanoscale resolution

  • Particularly useful for studying SDCBP2's association with membrane microdomains or nuclear structures

• Correlative light and electron microscopy (CLEM):

  • Combine fluorescence and electron microscopy

  • Provide ultrastructural context for SDCBP2 localization

Given SDCBP2's reported roles in both PIP2 binding and nuclear organization, special attention should be paid to its distribution between nuclear and cytoplasmic compartments. Validation data shows both nuclear and cytoplasmic localization patterns depending on cell type and experimental conditions .