STARD10 Antibody

What are the most common applications for STARD10 antibodies in research?

STARD10 antibodies are validated for multiple applications including Western Blotting (WB), Immunoprecipitation (IP), Immunohistochemistry (IHC), Immunocytochemistry (ICC), Immunofluorescence (IF), and ELISA. Most commercially available antibodies show reactivity with human, mouse, and rat samples, with some also reacting with rabbit, cow, dog, guinea pig, and horse samples . For optimal results, application-specific dilutions are recommended:

• Western Blot: 1:1000-1:8000

• Immunoprecipitation: 0.5-4.0 μg for 1.0-3.0 mg of total protein lysate

• Immunohistochemistry: 1:20-1:200

What is the molecular weight of STARD10 and how does this affect antibody selection?

STARD10 has a calculated molecular weight of 33 kDa (291 amino acids), but is typically observed at 35-40 kDa on Western blots . Some studies have detected duplicate bands between 35-40 kDa, which should be considered when interpreting results . When selecting antibodies, researchers should verify that validation data confirms detection at the appropriate molecular weight range.

How should STARD10 antibodies be stored to maintain optimal activity?

Most STARD10 antibodies should be stored at -20°C and are stable for one year after shipment. For antibodies supplied in liquid form with PBS containing 0.02% sodium azide and 50% glycerol at pH 7.3, aliquoting is often unnecessary for -20°C storage . Some preparations, especially those in smaller volumes (20 μL), may contain 0.1% BSA to enhance stability . Always verify storage recommendations provided by the manufacturer.

How should researchers optimize antigen retrieval for STARD10 immunohistochemistry?

For optimal IHC detection of STARD10 in human tissue samples such as liver, it is recommended to perform antigen retrieval with TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 may also be effective . Comparative testing of both retrieval methods is advisable when establishing the protocol for a new tissue type, as optimal conditions may vary between tissues.

What controls should be included when validating STARD10 antibodies for specific applications?

When validating STARD10 antibodies, researchers should include:

• Positive tissue/cell controls: MCF-7 cells, T-47D cells for Western blotting; HepG2 cells for immunoprecipitation; and human liver tissue for immunohistochemistry

• Negative controls: Tissues/cells known not to express STARD10 or isotype controls

• Knockdown/knockout validation: Samples from STARD10 knockout models (such as the β STARD10KO mice) or cells treated with STARD10 siRNA

• Peptide competition assays: Pre-incubation of antibody with immunizing peptide to confirm specificity

What are the key considerations when selecting between monoclonal and polyclonal STARD10 antibodies?

The selection between monoclonal and polyclonal STARD10 antibodies depends on the specific research requirements:

For detecting post-translational modifications, such as STARD10 phosphorylation by casein kinase II, polyclonal antibodies may offer advantages by recognizing multiple epitopes .

How can researchers effectively use STARD10 antibodies in studying type 2 diabetes mechanisms?

STARD10 has been identified as a potential mediator in type 2 diabetes, with risk alleles associated with decreased STARD10 expression in β-cells. For studying this pathway:

• Use selective β-cell knockout models (β STARD10KO mice) to recapitulate features observed in human carriers of risk alleles

• Combine antibody-based detection methods with lipidomic analyses to investigate alterations in phosphatidylinositol levels

• Employ co-immunoprecipitation with STARD10 antibodies to identify binding partners like PIP4K2C

• Analyze insulin processing and granule morphology through electron microscopy in conjunction with immunolabeling

Research has shown that β STARD10KO islets exhibit altered dense core granule appearance with increased "rod-like" dense cores and elevated basal proinsulin secretion, suggesting STARD10's role in insulin granule biogenesis and maturation .

What approaches can be used to study STARD10-ERBB2 cross-talk in breast cancer using antibodies?

To investigate the interplay between STARD10 and ERBB2 in breast cancer:

• Perform co-expression analysis using Western blotting with both STARD10 and ERBB2 antibodies in breast cancer cell lines and clinical specimens

• Use promoter reporter assays to examine transcriptional regulation, especially following ethanol administration which induces both STARD10 and ERBB2 expression

• Conduct siRNA knockdown experiments with:

  • STARD10 siRNA sequences (e.g., 5'-GCCCUAUCCUUUACGUCAtt-3' and 5'-GGAGUACCCUGAGGCUAUAtt-3')

  • ERBB2 siRNA (commercial options available)

• Analyze downstream signaling pathways by examining p65 nuclear translocation and binding to both ERBB2 and STARD10 promoters

These approaches can help elucidate how STARD10 and ERBB2 positively regulate each other's expression and function, particularly in the context of ethanol exposure in breast cancer .

What methodological approaches should be used to analyze STARD10's interaction with phosphoinositides?

To investigate STARD10's role in phosphoinositide binding and transport:

• Perform X-ray crystallography of purified STARD10 protein to resolve its structure (previously solved to 2.3 Å resolution)

• Conduct molecular docking studies to identify potential binding pockets for phosphoinositides

• Use lipid overlay assays to confirm binding to specific phosphoinositides, particularly those phosphorylated at the 3' position

• Analyze phosphoinositide levels in knockout models using lipidomic approaches

• Investigate the expression of phosphoinositide-binding proteins (e.g., Pirt, Synaptotagmin 1) in STARD10 knockout models using antibody-based detection methods

These methods have previously revealed that STARD10 influences membrane lipid composition and insulin granule biogenesis through phosphatidylinositide binding and transport .

How can researchers address non-specific binding when using STARD10 antibodies?

To minimize non-specific binding and improve signal-to-noise ratio:

• Optimize blocking conditions using 10% serum or BSA in buffer solutions

• For IF applications, ensure proper fixation with paraformaldehyde and permeabilization with 0.1% Triton X-100

• Consider using highly specific monoclonal antibodies like C-11, which is available in multiple conjugated forms (agarose, HRP, PE, FITC, and Alexa Fluor®)

• Perform peptide competition assays to confirm specificity, particularly with polyclonal antibodies

• Validate antibody specificity using knockout or knockdown models before proceeding with detailed analyses

What are the best strategies for analyzing STARD10 in different cellular compartments?

STARD10 functions in lipid transport between different cellular compartments. To analyze its subcellular localization:

• For immunofluorescence studies: Use paraformaldehyde fixation followed by Triton X-100 permeabilization to preserve cellular architecture

• For fractionation studies: Combine subcellular fractionation with Western blotting using STARD10 antibodies

• For co-localization analyses: Perform dual immunofluorescence with antibodies against STARD10 and compartment-specific markers

• For dynamic studies: Consider using fluorescently tagged STARD10 constructs (validated against antibody detection) for live-cell imaging

When studying sperm cells, where STARD10 localizes to the flagellum and may function in energy metabolism, special fixation and permeabilization protocols may be required for effective antibody penetration .

How can epitope-specific STARD10 antibodies be leveraged to study post-translational modifications?

Different STARD10 antibodies target specific regions of the protein:

• Internal region antibodies (e.g., ABIN6259482)

• C-terminal antibodies (e.g., ABIN2790346)

• Region-specific antibodies (e.g., AA 241-291)

To study post-translational modifications:

• Select antibodies that don't target known modification sites if studying the total STARD10 population

• Use modification-specific antibodies (e.g., phospho-specific) when available

• For phosphorylation studies, particularly STARD10 regulation by casein kinase II, combine immunoprecipitation with phospho-specific detection methods

• Compare results from multiple antibodies targeting different epitopes to gain comprehensive understanding of STARD10 regulation

What methodological approaches can address data discrepancies when using different STARD10 antibodies?

When faced with conflicting results using different STARD10 antibodies:

• Validate each antibody's specificity using knockout/knockdown controls and Western blotting

• Consider epitope availability issues that may affect antibody binding in different applications

• Test multiple antibodies targeting different regions of STARD10 in parallel

• Verify results using complementary techniques (e.g., mass spectrometry)

• Account for potential splice variants or post-translational modifications that might affect epitope recognition

• Document and report antibody catalog numbers, dilutions, and experimental conditions to ensure reproducibility

This systematic approach helps reconcile discrepancies and ensures reliable research findings when working with different STARD10 antibodies.

How can STARD10 antibodies be utilized in multi-omics approaches to understand lipid metabolism disorders?

For integrating STARD10 research into multi-omics studies:

• Combine immunoprecipitation using STARD10 antibodies with mass spectrometry to identify protein-protein interactions

• Integrate STARD10 protein expression data (from antibody-based assays) with transcriptomics and lipidomics datasets

• Use STARD10 antibodies for ChIP-seq to identify transcriptional networks regulated by STARD10-associated complexes

• Employ tissue microarrays with STARD10 immunohistochemistry to correlate expression with clinical parameters across patient cohorts

These approaches can provide comprehensive insights into STARD10's role in lipid metabolism disorders, particularly in diabetes and cancer contexts .

What are the considerations for using STARD10 antibodies in translational research for diabetes biomarker development?

When developing STARD10 as a potential biomarker for diabetes risk or progression:

• Validate antibody specificity against recombinant STARD10 and in clinical samples

• Establish standardized protocols for sample processing and antibody-based detection

• Correlate STARD10 levels with established diabetes markers and genetic risk factors

• Consider developing quantitative assays (ELISA, multiplexed platforms) using validated STARD10 antibodies

• Evaluate STARD10 expression in accessible tissues or liquid biopsies that might reflect β-cell dysfunction

Research has established that risk alleles for type 2 diabetes at the STARD10 locus are associated with lowered STARD10 expression in β-cells, impaired glucose-induced insulin secretion, and decreased circulating proinsulin:insulin ratios .

How can researchers design experimental systems to study STARD10's function in phosphoinositide-mediated signaling?

To investigate STARD10's role in phosphoinositide signaling:

• Develop in vitro lipid transfer assays using purified STARD10 protein and fluorescently labeled phosphoinositides

• Create cellular reporter systems for monitoring phosphoinositide levels in response to STARD10 manipulation

• Employ CRISPR/Cas9 genome editing to introduce specific mutations in STARD10's phosphoinositide binding pocket

• Establish reconstitution systems in STARD10-knockout cells to test the function of wildtype versus mutant STARD10