BCAS2 Antibody

What is BCAS2 and why is it significant in cancer research?

BCAS2, also known as pre-mRNA-splicing factor SPF27 or DAM1 (DNA amplified in mammary carcinoma 1 protein), is a nuclear protein that plays crucial roles in the spliceosome complex, which is responsible for removing introns from precursor mRNA. It ensures proper exon joining to form mature mRNA for protein translation . BCAS2 has gained significant research interest due to its overexpression in various cancers, particularly breast and prostate cancer, suggesting its involvement in cancer progression . Additionally, BCAS2 interacts with several key proteins, including estrogen receptors, and can function as a coactivator enhancing transcriptional activity, potentially contributing to carcinogenesis .

What are the recommended applications for BCAS2 antibodies in laboratory research?

BCAS2 antibodies have been validated for multiple experimental applications:

For optimal results, researchers should titrate the antibody concentration for their specific experimental system . When selecting an antibody, consider the species reactivity, clonality (polyclonal vs. monoclonal), and validated applications that align with your research objectives .

How should BCAS2 antibodies be stored and handled to maintain reactivity?

BCAS2 antibodies are typically supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3 . For optimal stability:

• Store antibodies at -20°C for long-term storage (stable for up to one year)

• Store at 4°C for short-term use (up to three months)

• Avoid repeated freeze-thaw cycles that can degrade antibody quality

• Consider aliquoting the antibody solution into smaller volumes upon receipt

• Keep antibodies away from prolonged exposure to high temperatures

• Follow manufacturer's specific recommendations for reconstitution if supplied in lyophilized form

How should researchers validate BCAS2 antibody specificity for their experimental systems?

Antibody validation is critical to ensure experimental rigor:

• Positive and negative controls: Use tissues/cells known to express or lack BCAS2 (positive in HeLa, MCF-7, mouse brain; negative in BCAS2 knockout samples)

• Multiple antibody approach: Employ antibodies from different vendors or those recognizing different epitopes

• Knockdown/knockout validation:

  • Perform siRNA knockdown or CRISPR knockout of BCAS2

  • Confirm reduced signal in Western blot or immunostaining

  • Example: In LNCaP and PC-3 cell lines, shRNA-mediated BCAS2 knockdown showed decreased protein levels, validating antibody specificity

• Immunoprecipitation followed by mass spectrometry: Confirm target identity

• Peptide competition assay: Pre-incubate antibody with immunizing peptide to block specific binding

For publications, include detailed validation data to strengthen the reliability of your findings.

What are the key considerations for detecting BCAS2 in tissue samples via immunohistochemistry?

When performing IHC with BCAS2 antibodies, consider these methodological factors:

• Tissue preparation:

  • Fix tissues with 4% paraformaldehyde for 48 hours

  • Process through gradient ethanol dehydration, xylene transparency, and paraffin embedding

  • Section at 5 μm thickness

• Antigen retrieval:

  • Use TE buffer (pH 9.0) as the primary recommendation

  • Alternative: citrate buffer (pH 6.0)

  • Apply microwave heating for optimal results

• Antibody dilution:

  • Start with 1:50-1:500 dilution range

  • Optimize based on tissue type and detection system

• Signal development:

  • For colorimetric detection, use DAB kit

  • Counterstain with hematoxylin

  • For fluorescence detection, use appropriate secondary antibodies (e.g., FITC-labeled)

• Positive controls:

  • Human breast cancer tissue shows strong BCAS2 expression

  • Human prostate cancer tissue also shows high expression

What are the molecular weight considerations when detecting BCAS2 via Western blot?

When analyzing BCAS2 protein by Western blot:

• Expected molecular weight:

  • Calculated molecular weight: 26 kDa

  • Observed molecular weight range: 26-32 kDa

  • Some sources report observation at ~68 kDa in certain conditions

• Sample preparation:

  • Use RIPA buffer with PMSF for protein extraction

  • Denature samples at 95°C for 10 minutes

  • Load 20 μg of protein per lane on 12% SDS-PAGE gels

• Transfer conditions:

  • Wet transfer to PVDF membrane

  • 100V for 90 minutes

• Antibody incubation:

  • Primary antibody dilution: 1:1000-1:8000

  • Overnight incubation at 4°C

  • Secondary antibody incubation: 2 hours at room temperature

• Detection system:

  • Enhanced chemiluminescence (ECL) for visualization

  • Band quantification relative to loading control (β-actin, GAPDH)

The presence of multiple bands or unexpected molecular weights should be carefully validated to distinguish between isoforms, post-translational modifications, and non-specific binding.

How can researchers effectively use BCAS2 antibodies to study DNA double-strand break (DSB) repair mechanisms?

BCAS2 is involved in DNA double-strand break repair through both non-homologous end joining (NHEJ) and homologous recombination (HR) pathways . For studying these mechanisms:

• Radiation-induced DSB experiments:

  • Treat cells with ionizing radiation to induce DSBs

  • Use BCAS2 antibody alongside γ-H2AX antibody (DSB marker)

  • Monitor BCAS2 localization and γ-H2AX foci resolution

  • Example: BCAS2 knockdown in LNCaP and PC-3 cells showed increased γ-H2AX levels after radiation, indicating impaired DSB repair

• Co-immunoprecipitation with DSB repair proteins:

  • Use BCAS2 antibody to pull down protein complexes

  • Probe for interaction partners like NBS1, which is involved in both NHEJ and HR

  • Example: BCAS2 interacts with NBS1 via its N-terminus and both the N- and C-termini of NBS1

• Chromatin immunoprecipitation (ChIP):

  • Map BCAS2 recruitment to DSB sites using BCAS2 antibodies

  • Combine with next-generation sequencing (ChIP-seq) for genome-wide analysis

• Immunofluorescence microscopy for repair foci:

  • Co-stain for BCAS2 and established DSB markers

  • Monitor BCAS2 recruitment to damage sites over time

When interpreting results, consider that BCAS2's role in DSB repair may be context-dependent and influenced by cell type and p53 status .

What methodological approaches help investigate BCAS2's role in pre-mRNA splicing using specific antibodies?

To study BCAS2's function in pre-mRNA splicing:

• Crosslinking immunoprecipitation and sequencing (CLIP-seq):

  • Crosslink RNA-protein complexes in vivo

  • Immunoprecipitate with BCAS2 antibody

  • Sequence bound RNA fragments

  • Example: CLIP-seq revealed BCAS2 predominantly binds to 5' splice sites of introns and shows preference for GA-rich regions

• RNA immunoprecipitation (RIP):

  • Use BCAS2 antibody to immunoprecipitate RNA-protein complexes

  • Analyze bound RNAs by RT-PCR or sequencing

  • Example: RIP-qPCR validated BCAS2 binding to pre-mRNAs of genes involved in DNA repair and synapsis

• Co-immunoprecipitation with spliceosome components:

  • Immunoprecipitate with BCAS2 antibody

  • Probe for interaction with other splicing factors

  • Example: BCAS2 interacts with PRP19 and CDC5L, key components of the spliceosome

• Alternative splicing analysis:

  • Compare splicing patterns in BCAS2 knockdown/knockout vs. control cells

  • Use RT-PCR to validate specific splicing events

  • Example: BCAS2 deficiency led to abnormal alternative splicing of Trp53bp1 (coding for 53BP1) and Six6os1

These approaches can help uncover both global and gene-specific roles of BCAS2 in mRNA processing.

How can researchers investigate the association between BCAS2 and hormone receptors in cancer progression?

BCAS2 interacts with multiple hormone receptors and may contribute to cancer development. To study these interactions:

• GST pull-down assays:

  • Use GST-tagged hormone receptor constructs (full-length and domains)

  • Pull down biotin-labeled in vitro translated BCAS2

  • Example: GST pull-down showed BCAS2 interaction with estrogen receptor alpha (ERα) in both presence and absence of estradiol (E2)

• Co-immunoprecipitation:

  • Immunoprecipitate with BCAS2 antibody from cancer cell lysates

  • Probe for hormone receptors (ERα, PR, etc.)

  • Test hormone-dependence by treating cells with ligands before IP

  • Example: Co-IP in MCF7 cells showed BCAS2-ERα interaction in the presence of estradiol or tamoxifen

• Luciferase reporter assays:

  • Transfect cells with hormone-responsive reporter constructs

  • Co-transfect BCAS2 expression plasmid

  • Measure transcriptional activity with/without hormone treatment

  • Example: BCAS2 enhanced ERα-mediated transcription in MCF7, MDA-MB-231, and SK-BR-3 cells

• RT-qPCR for target genes:

  • Overexpress or knockdown BCAS2 in hormone-responsive cells

  • Measure expression of known hormone-regulated genes

  • Example: BCAS2 overexpression in MCF7 cells increased expression of estrogen-responsive genes like pS2, C3, IGFBP2, and C-MYC

• Immunohistochemistry in tumor tissues:

  • Use BCAS2 antibody alongside hormone receptor antibodies

  • Analyze correlation between expression patterns

  • Example: BCAS2 overexpression was associated with higher Gleason grade in prostate cancer

What are common issues when using BCAS2 antibodies, and how can researchers address them?

When working with BCAS2 antibodies, researchers may encounter several challenges:

• High background in immunostaining:

  • Optimize blocking (try 5% BSA or skimmed milk)

  • Increase washing steps (3-5 times with TBST)

  • Titrate antibody to lower concentration

  • Use more specific secondary antibodies

  • Include additional blocking steps with avidin/biotin for biotinylated detection systems

• Multiple bands in Western blot:

  • Verify sample preparation (complete denaturation)

  • Check for protein degradation (add protease inhibitors)

  • Consider post-translational modifications

  • Use more stringent washing conditions

  • Perform peptide competition assay to identify specific bands

• Weak or no signal:

  • Check protein expression levels in your sample

  • Optimize antigen retrieval for IHC/IF

  • Increase antibody concentration or incubation time

  • Use more sensitive detection systems (enhanced ECL)

• Non-reproducible results:

  • Standardize protocols across experiments

  • Prepare fresh working solutions

  • Document lot-to-lot variations

  • Consider using different antibody clones

How can researchers optimize BCAS2 immunoprecipitation protocols for protein interaction studies?

For effective BCAS2 immunoprecipitation:

• Cell/tissue lysis optimization:

  • For tissues: Homogenize thoroughly in appropriate buffer

  • For cells: Use gentle lysis conditions to preserve protein complexes

  • Include phosphatase inhibitors for studying phosphorylation-dependent interactions

• Pre-clearing step:

  • Incubate lysate with protein A/G beads before adding antibody

  • Remove non-specific binding proteins

• Antibody binding:

  • Use 0.5-4.0 μg antibody per 1.0-3.0 mg of protein lysate

  • Include IgG control for non-specific binding assessment

  • Pre-adsorb antibody to protein A/G magnetic beads

• Washing optimization:

  • Balance between stringency (to reduce background) and maintaining interactions

  • Typically use 3-5 washes with TBST

  • Consider salt or detergent gradients for specific applications

• Elution and detection:

  • Gentle elution for maintained interactions

  • Boil in SDS loading buffer for complete dissociation

  • Western blot with specific antibodies for interaction partners

Example protocol: For studying BCAS2 interaction with splicing factors, eight-day-old ovaries were solubilized in cell lysis buffer, followed by antibody adsorption using BeyoMag™ Protein A. After overnight incubation at 4°C, beads were washed three times with TBST and boiled in 1× SDS loading buffer for Western blot analysis .

What cellular compartments show BCAS2 localization, and how should researchers optimize immunofluorescence studies?

BCAS2 primarily localizes to the nucleus, particularly in regions associated with splicing and DNA repair. For optimal immunofluorescence studies:

• Fixation optimization:

  • For cultured cells: 4% paraformaldehyde for 15-20 minutes

  • For tissue sections: Follow paraffin section preparation protocols

• Permeabilization considerations:

  • Nuclear localization requires effective permeabilization

  • 0.1-0.5% Triton X-100 is typically effective

  • Balance permeabilization with epitope preservation

• Antigen retrieval for tissues:

  • EDTA antigen retrieval buffer with microwave heating

  • Critical for detecting nuclear antigens in formalin-fixed tissues

• Antibody concentration:

  • Start with 1:400-1:1600 for cultured cells

  • Higher concentrations (1:50-1:400) may be needed for tissue sections

• Counterstaining options:

  • DAPI for nuclear visualization

  • Consider co-staining with other nuclear markers (splicing factors, DNA repair proteins)

• Mounting and imaging:

  • Use anti-fade mounting medium to prevent photobleaching

  • Confocal microscopy for precise localization studies

Positive controls such as HeLa and MCF-7 cells show clear nuclear BCAS2 staining and can be used to validate immunofluorescence protocols .

How can BCAS2 antibodies be utilized in studying reproductive biology and fertility research?

Recent studies have revealed BCAS2's importance in reproductive biology:

• Spermatogenesis research:

  • Use immunohistochemistry with BCAS2 antibodies to examine expression in testicular development

  • Example: BCAS2 is critical for testicular development and spermatogenesis in Hezuo pig, potentially by regulating cell proliferation or differentiation

• Conditional knockout models:

  • Use BCAS2 antibodies to validate knockout efficiency

  • Examine phenotypes related to fertility

  • Example: Conditional knockout of Bcas2 in mouse germ cells resulted in impaired DSB repair and synapsis during meiosis, leading to non-obstructive azoospermia (NOA)

• Meiotic processes:

  • Immunofluorescence to study BCAS2 localization during meiotic prophase

  • Co-localization with meiotic markers

  • Example: BCAS2 regulates alternative splicing of genes crucial for meiosis, including Spo11, Six6os1, Sycp1, Msh5, Cdk2, Sun2, Stag3, and Spdya

• Female fertility studies:

  • Examine BCAS2 in granulosa cell function

  • Example: BCAS2 influences proliferation and survival of granulosa cells through regulating pre-mRNA splicing of E2f3 and Flt3l

These applications highlight BCAS2's broader biological significance beyond cancer research.

What are the advanced approaches for using BCAS2 antibodies in cancer biomarker research?

For investigating BCAS2 as a potential cancer biomarker:

• Tissue microarray analysis:

  • Apply BCAS2 antibodies to tissue microarrays from patient cohorts

  • Correlate expression with clinical parameters and outcomes

  • Example: BCAS2 overexpression was significantly associated with higher Gleason and pathology grades and shorter survival in prostate cancer patients

• Multiplex immunohistochemistry/immunofluorescence:

  • Combine BCAS2 antibody with other cancer markers

  • Use spectral unmixing for co-localization analysis

  • Example: BCAS2 can be detected alongside hormone receptors in breast cancer samples

• Circulating tumor cell (CTC) analysis:

  • Apply immunofluorescence for BCAS2 in liquid biopsies

  • Evaluate potential as a blood-based biomarker

• Proximity ligation assay (PLA):

  • Detect protein-protein interactions in situ

  • Study BCAS2 interactions with oncogenic partners

  • Example: BCAS2-ERα interactions could be visualized in breast cancer tissues

• Chromatin immunoprecipitation with sequencing (ChIP-seq):

  • Map genomic binding sites of BCAS2 in cancer cells

  • Identify target genes regulated by BCAS2

These approaches can help determine whether BCAS2 has potential as a diagnostic, prognostic, or predictive biomarker in different cancer types.

How can researchers integrate BCAS2 antibody-based studies with genomic and transcriptomic data?

To create comprehensive multi-omics analyses involving BCAS2:

• Integration with RNA-seq:

  • Correlate BCAS2 protein levels (detected by antibodies) with alternative splicing events

  • Example: BCAS2 deficiency led to abnormal alternative splicing patterns visible in RNA-seq data, which were validated by RT-PCR

• Combining ChIP-seq with proteomics:

  • Use BCAS2 antibodies for ChIP-seq to identify genomic binding sites

  • Correlate with protein interaction data from immunoprecipitation studies

  • Example: BCAS2's role in transcriptional regulation can be linked to its binding partners, such as hormone receptors

• Validation of genetic findings with protein analysis:

  • Confirm the effects of genetic variants on BCAS2 protein expression or localization

  • Use antibodies to determine if mutations affect protein-protein interactions

• Single-cell approaches:

  • Combine single-cell RNA-seq with immunofluorescence

  • Correlate BCAS2 protein levels with transcriptional profiles at the single-cell level

• Functional validation of computational predictions:

  • Use antibody-based methods to validate predictions from bioinformatic analyses

  • Example: BCAS2's predicted binding to specific pre-mRNAs was validated through RNA immunoprecipitation with BCAS2 antibodies

This integrative approach provides a more comprehensive understanding of BCAS2's functions and regulatory networks in both normal and disease states.