BCAS1 Antibody

What is BCAS1 and why is it significant in research?

BCAS1 was initially identified through positional cloning and found to be overexpressed in most breast cancer cell lines. It is a cytoplasmic protein that forms homodimers through coiled-coil structures. Its significance spans multiple research areas, with particularly high expression in brain and prostate tissues, and lower amounts in colon, intestine, and testis . Recent research has revealed that BCAS1 serves as a critical marker for newly formed, myelinating oligodendrocytes in both mouse and human brains, marking regions of active myelin formation during development and in adulthood .

What applications are BCAS1 antibodies typically used for?

BCAS1 antibodies have been validated for multiple experimental applications:

The specific conditions for optimal reactivity should be determined by the end user, as sensitivity varies between antibody sources and experimental systems .

How should BCAS1 antibodies be stored and handled?

BCAS1 antibodies are typically supplied in liquid form with stabilizing buffers. Optimal storage conditions include:

• Temperature: Most BCAS1 antibodies should be stored at 4°C for short-term use (up to one year) or at -20°C for long-term storage .

• Buffer conditions: Commonly stored in PBS with sodium azide (0.02%) and glycerol (50%), pH 7.2-7.3 .

• Critical handling note: Antibodies should not be exposed to prolonged high temperatures as this may result in degradation and loss of activity .

• Many suppliers recommend aliquoting to avoid repeated freeze-thaw cycles for antibodies stored at -20°C .

How should researchers validate a BCAS1 antibody for their specific experimental system?

Validation should follow a multi-step approach:

• Positive and negative controls: Use tissues or cell lines known to express or lack BCAS1. Human lung tissue lysate, NIH/3T3 cells, and MCF-7 cells serve as good positive controls .

• Specificity verification:

  • Preabsorption of the antibody with recombinant BCAS1 protein before immunolabeling should eliminate staining .

  • Transfection of BCAS1 cDNA into cells followed by immunofluorescence should show labeling only in transfected cells .

  • Use of Bcas1 knockout mouse tissue should show absence of staining .

  • Co-staining with multiple BCAS1 antibodies (e.g., polyclonal antiserum with monoclonal antibody) should show overlapping patterns .

• Molecular weight confirmation: In Western blots, confirm the expected molecular weight (61-68 kDa depending on the source and modifications) .

What are the best experimental conditions for BCAS1 antibody in immunohistochemistry?

For optimal IHC results with BCAS1 antibodies:

• Tissue preparation: Use frozen tissue sections or properly fixed paraffin-embedded sections.

• Antigen retrieval:

  • TE buffer at pH 9.0 is recommended for many BCAS1 antibodies

  • Alternatively, citrate buffer at pH 6.0 may be used .

• Antibody concentration: Start with dilutions between 1:50-1:500 and optimize for your specific tissue .

• Detection systems: Both chromogenic and fluorescent detection systems are compatible with BCAS1 antibodies. For co-localization studies, immunofluorescence is preferable .

• Controls: Include a negative control (omitting primary antibody) and positive control tissue known to express BCAS1 (e.g., human stomach tissue) .

How can BCAS1 antibodies be used to study oligodendrocyte development?

BCAS1 antibodies provide a unique tool for studying oligodendrocyte development because:

• Marker for specific developmental stage: BCAS1 expression identifies a transient population of newly formed, myelinating oligodendrocytes that segregate from both oligodendrocyte progenitor cells and mature oligodendrocytes .

• Experimental approach:

  • Use BCAS1 antibodies in combination with markers for oligodendrocyte lineage (SOX10, OLIG1, OLIG2)

  • Co-stain with progenitor cell markers (NG2) and mature oligodendrocyte markers (CC1, MAG) to distinguish developmental stages

  • In mouse studies, approximately 16.5% of BCAS1+ cells are oligodendrocyte progenitor cells, while the remaining are more differentiated .

• Time course studies: BCAS1 expression is developmentally regulated and gradually decreases as oligodendrocytes mature, making it valuable for time course studies of myelination .

How do BCAS1+ oligodendrocytes differ between development and adulthood?

BCAS1 expression patterns change significantly between developmental stages and adulthood:

• Developmental expression:

  • In early postnatal development, BCAS1+ oligodendrocytes are abundant in white matter regions

  • A subpopulation of premyelinating oligodendrocytes express both BCAS1 and PLP during development (29.5% at P9, 23.7% at P14) .

• Adult expression:

  • In adult mice (4 months), BCAS1+ premyelinating oligodendrocytes are negative for PLP

  • Complete segregation of GFP+ (PLP marker) and BCAS1+ myelinating oligodendrocytes occurs in adulthood

  • Only about 4% of fully mature MBP+ oligodendrocytes express BCAS1 .

• Regional differences:

  • In humans, high density of BCAS1+ oligodendrocytes is found only in white matter during fetal and early postnatal periods

  • In the frontal cortex, BCAS1+ oligodendrocytes remain numerous throughout adulthood .

What methodological approaches can be used to study BCAS1 during myelination and remyelination?

Several approaches are particularly effective:

• In vitro cultures:

  • Primary oligodendrocyte cultures show BCAS1 expression in 85.9% of O4+ oligodendrocytes with arborized morphology at 3 days in vitro

  • BCAS1 expression decreases after 5 days when cells form myelin sheets and express MBP

  • BCAS1 becomes excluded from compact myelin-like membrane sheets but remains at low levels in non-compacted areas .

• In vivo demyelination models:

  • BCAS1 antibodies can identify active remyelination in animal models

  • In multiple sclerosis (MS) patient samples, BCAS1+ oligodendrocytes at lesion edges suggest active endogenous remyelination even at late disease stages .

• Human iPSC-derived oligodendrocytes:

  • BCAS1 antibodies can be used to identify newly formed oligodendrocytes in cultures derived from induced pluripotent stem cells

  • This provides a human-relevant model for studying myelination processes .

How can BCAS1 antibodies be utilized in breast cancer research?

BCAS1 antibodies offer several methodological approaches for cancer research:

• Expression analysis:

  • Western blotting to quantify BCAS1 protein levels across different breast cancer cell lines and tissue samples

  • Immunohistochemistry to examine heterogeneity of BCAS1 expression within tumors and correlate with clinical parameters

• Functional studies:

  • Although cells engineered to overexpress BCAS1 did not lose anchorage-dependent growth or increase growth rate (suggesting it's not a prototypical oncogene), BCAS1 may still play important roles in cancer biology .

  • Knockdown/knockout studies combined with BCAS1 antibody validation can help establish the functional significance of BCAS1 in cancer cells.

• Amplification detection:

  • Combined approaches using FISH for gene amplification and antibody-based methods for protein detection can provide insights into the relationship between BCAS1 gene amplification and protein expression .

What experimental controls are essential when investigating BCAS1 in pancreatic carcinoma?

When studying BCAS1 in pancreatic carcinoma:

• Tissue controls:

  • Normal pancreatic tissue adjacent to tumor

  • Pancreatic developmental tissue series to understand expression changes

  • Panel of pancreatic cancer cell lines with varying levels of BCAS1 expression

• Experimental controls:

  • Antigen competition assays to verify antibody specificity in pancreatic tissues

  • BCAS1 knockdown/overexpression in pancreatic cancer cells to validate antibody detection

  • Correlation with genomic data to confirm relationship between gene amplification and protein expression

• Methodological approach:

  • Multi-antibody validation using both polyclonal and monoclonal antibodies against different epitopes

  • Combination of techniques (IHC, WB, IF) to confirm expression patterns

  • Quantitative analysis methods to determine threshold levels relevant to pathological significance .

How should researchers address non-specific staining issues with BCAS1 antibodies?

Non-specific staining can be addressed through several methodological approaches:

• Antibody optimization:

  • Titrate antibody concentrations (start with recommended dilutions of 1:200-1:1000 for WB, 1:50-1:500 for IHC, and 1:200-1:800 for IF) .

  • Optimize incubation time and temperature

  • Consider different blocking reagents (BSA, normal serum, commercial blockers)

• Validation controls:

  • Preabsorption with BCAS1 peptide (e.g., PEP-1018 for PA5-20904)

  • Use of Bcas1 knockout tissue as negative control

  • Secondary antibody-only controls to detect non-specific binding

• Technical modifications:

  • Adjust fixation methods and duration

  • Try different antigen retrieval methods (TE buffer pH 9.0 vs. citrate buffer pH 6.0)

  • Increase washing steps and duration

  • For problematic tissues, consider tissue-specific method optimization

What are the most effective co-staining approaches for BCAS1 in oligodendrocyte lineage studies?

For co-staining experiments investigating oligodendrocyte development:

• Recommended marker combinations:

  • BCAS1 + lineage markers (SOX10, OLIG1, OLIG2) to confirm oligodendrocyte identity

  • BCAS1 + NG2 to identify the subset of BCAS1+ cells that are oligodendrocyte progenitors (approximately 16.5%)

  • BCAS1 + CC1 (adenomatous polyposis coli antibody) to identify more differentiated cells (76.2% overlap)

  • BCAS1 + MAG to identify cells involved in oligodendrocyte-axon contact (47.5% overlap)

  • BCAS1 + MBP to distinguish from fully mature oligodendrocytes (only 4% overlap)

• Technical considerations:

  • Ensure primary antibodies are raised in different host species to avoid cross-reactivity

  • Sequential staining may be necessary for challenging combinations

  • Use of directly conjugated antibodies can minimize background in multi-color experiments

  • Confocal microscopy is recommended for accurate co-localization analysis

• Quantification methods:

  • Cell counting in multiple fields using unbiased stereological approaches

  • Fluorescence intensity correlation for protein expression levels

  • Morphological classification combined with marker expression

How might BCAS1 antibodies contribute to multiple sclerosis research?

BCAS1 antibodies offer unique advantages in MS research:

• Identification of remyelination:

  • BCAS1+ oligodendrocytes have been observed at lesion edges in a proportion of fully demyelinated white matter lesions in MS patients

  • This suggests endogenous remyelination might be active during MS even at late stages

• Methodological approach:

  • Use BCAS1 antibodies to quantify newly formed oligodendrocytes in MS tissue samples

  • Correlate BCAS1+ cell numbers with clinical parameters and disease progression

  • Combine with other markers to assess the full oligodendrocyte lineage in lesions

• Therapeutic implications:

  • BCAS1 antibodies could be used to evaluate the efficacy of remyelination-promoting therapies

  • Monitoring BCAS1+ cell populations in response to treatment could provide a cellular readout of remyelination potential

What considerations are important when designing experiments to study BCAS1 in neurodevelopmental disorders?

When investigating BCAS1 in neurodevelopmental contexts:

• Developmental timing:

  • BCAS1 expression is temporally regulated, so precise developmental staging is critical

  • Design experiments to capture the transient nature of BCAS1 expression in oligodendrocyte lineage

  • Include multiple time points spanning development and maturation

• Regional specificity:

  • BCAS1 expression patterns differ between brain regions

  • High density of BCAS1+ oligodendrocytes is found in white matter only during fetal and early postnatal periods

  • In frontal cortex, BCAS1+ oligodendrocytes remain numerous throughout adulthood

• Methodological approaches:

  • Combine in vivo studies with in vitro models (primary cultures, iPSC-derived oligodendrocytes)

  • Consider single-cell approaches to capture heterogeneity within BCAS1+ populations

  • Correlate BCAS1 expression with functional measures of myelination and circuit development