basp1 Antibody

What is BASP1 and why is its molecular weight so variable in experimental data?

BASP1 is a signaling protein that plays key roles in neurite outgrowth and plasticity . While its calculated molecular weight is 23 kDa, it is frequently observed at 55-60 kDa in Western blot analyses . This discrepancy occurs due to extensive post-translational modifications, including possible SUMOylation, as evidenced by research detecting a specific band at approximately 60 kDa . The protein exists in multiple isoforms, with both higher and lower molecular weight variants observed in human cell lines .

What is the expression pattern of BASP1 across different tissues and developmental stages?

BASP1 shows distinctive temporal and spatial expression patterns:

• During development: Expressed throughout the brain

• In adulthood: Expression becomes restricted to neurogenic regions

• In adult hippocampal niche: Limited to type I radial neural stem cells (NSCs)

• In subventricular zone (SVZ): Present in B and C cells and GFAP-expressing cells in the rostral migratory stream

• In cancer: Upregulated in head and neck squamous cell carcinoma (HNSCC) and associated with poor prognosis

The NSC-6 antibody specifically recognizes BASP1 in neurogenic regions of postnatal mouse brain, including the corpus callosum, anterior commissure, cerebellum (in Bergmann glia radial processes), and the dentate gyrus of the hippocampus .

How do I select the most appropriate BASP1 antibody for my specific research application?

When selecting a BASP1 antibody, consider these critical factors:

• Epitope specificity: Different antibodies target distinct regions of BASP1:

  • N-terminal region (e.g., Gly2-Ala45)

  • Middle region (e.g., AA 129-159)

  • C-terminal region

• Validated applications: Verify the antibody has been validated for your specific application:

  • Western blot: Most BASP1 antibodies have been validated

  • Immunohistochemistry: Consider antibodies with demonstrated specificity in tissue sections

  • Immunofluorescence: Evaluate background levels and specific staining patterns

  • Flow cytometry: Some antibodies are specifically validated for intracellular flow cytometry

• Species reactivity: Confirm cross-reactivity with your species of interest:

  • Human: Most commercially available antibodies

  • Mouse: Selected antibodies

  • Other species: Limited validation data available

• Clonality: Consider the advantages of each format:

  • Polyclonal: Higher sensitivity but potentially more background

  • Monoclonal: Higher specificity and reproducibility

What validation methods should I employ to confirm BASP1 antibody specificity?

Rigorous validation is essential for generating reliable data with BASP1 antibodies:

• RNAi validation: The gold standard approach involves comparing antibody staining in control cells versus cells where BASP1 has been knocked down using siRNA. Western blot data shows successful validation using this approach for multiple antibodies .

• Positive control samples: Use tissues or cell lines with confirmed BASP1 expression:

  • HeLa cells: Consistently express BASP1

  • DU145 cells: Prostate carcinoma cell line with BASP1 expression

  • A549 cells: Lung carcinoma cells with detectable BASP1

  • Human tonsil: Shows positive BASP1 staining in IHC

• Negative control samples: Include samples known to lack BASP1 expression:

  • K-562 cells: Chronic myelogenous leukemia lymphoblasts with minimal BASP1 expression

  • Human skeletal muscle: Shows negative staining with some BASP1 antibodies

• Immunoprecipitation-Western blot: Confirm specificity by immunoprecipitating BASP1 from cell lysates followed by Western blotting with the same or different BASP1 antibody .

What are the optimal conditions for BASP1 detection by Western blotting?

For reliable BASP1 detection by Western blotting, follow these optimized protocol parameters:

For optimal resolution, use reducing conditions with longer run times to separate potential isoforms .

What are the recommended protocols for BASP1 immunohistochemistry?

For successful BASP1 immunohistochemistry:

• Tissue preparation: Standard formalin fixation and paraffin embedding works well for most tissues .

• Antigen retrieval: Two effective methods:

  • Preferred: Tris-EDTA buffer (pH 9.0) with heat-mediated retrieval

  • Alternative: Citrate buffer (pH 6.0) if Tris-EDTA yields high background

• Blocking and antibody application:

  • Blocking: 5% normal serum from secondary antibody host species

  • Primary antibody dilution: 1:800 to 1:3200 (antibody-dependent)

  • Incubation time: 30 minutes at room temperature or overnight at 4°C

• Detection systems:

  • Polymer-based detection systems like Bond™ Polymer Refine Detection yield excellent results

  • DAB chromogen provides good contrast for brightfield microscopy

  • For fluorescence, use fluorophore-conjugated secondary antibodies with minimal cross-reactivity

• Controls:

  • Positive control: Human tonsil shows consistent BASP1 expression

  • Negative control: Human skeletal muscle shows minimal staining

How can I track BASP1 localization changes during cellular processes like apoptosis?

BASP1 undergoes significant relocalization during apoptosis, moving from the nucleus to the cytoplasm after caspase activation . To track this process:

• Dual immunofluorescence staining:

  • Co-stain for BASP1 and apoptotic markers (cleaved caspase-3, PARP)

  • Use confocal microscopy for precise subcellular localization

• Time-course experiments:

  • Induce apoptosis with standard triggers (staurosporine, FasL, UV)

  • Fix cells at multiple timepoints (0, 2, 4, 6, 12, 24 hours)

  • Quantify nuclear-to-cytoplasmic BASP1 ratio changes

• Live-cell imaging:

  • Express fluorescently-tagged BASP1 in cell lines

  • Monitor relocalization in real-time during apoptosis induction

  • Correlate with other apoptotic events using appropriate markers

• Biochemical fractionation:

  • Separate nuclear and cytoplasmic fractions at various timepoints

  • Perform Western blot analysis for BASP1 in each fraction

  • Quantify changing distribution patterns

Research suggests that the 9B1 antibody preferentially labels the cytoplasmic form of BASP1 in apoptotic cells, making it a potential tool for specifically detecting cells committed to apoptosis rather than those merely exhibiting caspase activation .

Why am I detecting multiple bands of BASP1 at unexpected molecular weights?

Multiple BASP1 bands on Western blots are common and can be attributed to several factors:

• Post-translational modifications:

  • SUMOylation: Can increase apparent molecular weight to 60 kDa

  • Other modifications: Phosphorylation, glycosylation may alter migration

• Multiple isoforms: BASP1 exists in different forms with varying expression across tissues and developmental stages

• Degradation products: Incomplete protease inhibition during sample preparation can lead to proteolysis

• Antibody specificity: Different antibodies targeting different epitopes may recognize distinct subsets of BASP1 forms

How can I optimize BASP1 detection in neural stem cells for neurogenesis studies?

BASP1 serves as a valuable marker for neural stem cells in neurogenic niches . To optimize detection:

• Fixation optimization:

  • For brain sections: 4% PFA fixation for 24 hours yields optimal results

  • For cultured NSCs: Brief fixation (10-15 minutes) with 4% PFA preserves epitopes

• Antigen retrieval assessment:

  • Test both Tris-EDTA (pH 9.0) and citrate buffer (pH 6.0) to determine optimal approach

  • Extend retrieval time (20-30 minutes) for fixed brain tissues

• Multi-marker co-localization:

  • In the hippocampal niche: Combine BASP1 staining with markers for type I radial NSCs

  • In the SVZ: Co-stain with markers for B and C cells to distinguish population subtypes

• Developmental comparisons:

  • Compare staining patterns across developmental timepoints

  • Note that BASP1 expression becomes restricted to neurogenic regions in adulthood

• Technical considerations:

  • Use thin sections (10-20 μm) for better antibody penetration

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

  • Include lipid permeabilization step for better antibody access

How can I use BASP1 antibodies to investigate its role in cancer immunotherapy response?

BASP1 has emerged as a potential predictor of immunotherapy response in head and neck squamous cell carcinoma (HNSCC) . To investigate this relationship:

• Expression analysis in patient cohorts:

  • Quantify BASP1 expression by IHC in tumor samples

  • Correlate with response to immune checkpoint inhibitors (ICIs)

  • Research shows BASP1-high patients exhibit better response rates to anti-PD-1 therapy

• Immune checkpoint correlation studies:

  • Co-stain for BASP1 and immune checkpoint molecules (PD-1, PD-L1, CTLA-4)

  • BASP1-high patients show higher expression of immune checkpoint inhibitors

• Tumor microenvironment assessment:

  • Analyze immune cell infiltration patterns in BASP1-high vs. BASP1-low tumors

  • Research indicates decreased activated CD8+ T cells in BASP1-high patients

• Mechanistic investigations:

  • Study BASP1's relationship with T cell dysfunction and immune escape

  • BASP1 is positively correlated with these processes

• Functional validation:

  • Perform BASP1 knockdown experiments in cancer cell lines

  • Assess changes in immunogenicity and response to immunotherapy

What methods can I use to study the relationship between BASP1 and ferroptosis?

Recent research has uncovered a novel connection between BASP1 and ferroptosis in cancer . To investigate this relationship:

• BASP1 modulation approaches:

  • siRNA knockdown: Use validated siRNAs targeting BASP1

  • Overexpression: Generate stable cell lines with BASP1 overexpression

  • CRISPR/Cas9: Create BASP1 knockout cell lines

• Ferroptosis marker assessment:

  • Lipid peroxidation: Use BODIPY-C11 or MDA assays

  • Glutathione measurement: Quantify reduced GSH levels

  • ROS detection: Use CM-H2DCFDA or similar ROS-sensitive probes

  • Cell death: Measure LDH release and viability

• Experimental design:

  • Compare BASP1-high vs. BASP1-low cells treated with:

    • Ferroptosis inducers (erastin, RSL3)

    • Ferroptosis inhibitors (ferrostatin-1, liproxstatin-1)

  • Research shows silencing BASP1 triggered ferroptosis in HNSCC cells

• Mechanistic pathway analysis:

  • Assess the expression of ferroptosis regulatory genes by qPCR

  • Examine protein expression of key ferroptosis regulators by Western blot

  • Investigate lipid metabolism alterations

How can I utilize BASP1 antibodies in developmental neuroscience and neural stem cell research?

BASP1 serves as a valuable marker for neural stem cells in neurogenic niches . To leverage this in research:

• Lineage tracing studies:

  • Use BASP1 antibodies to identify NSCs in the subgranular zone and subventricular zone

  • Combine with proliferation markers (Ki67, BrdU) to track neurogenesis

• Comparative developmental analysis:

  • Track BASP1 expression changes from embryonic to postnatal stages

  • BASP1 is widely expressed throughout the brain during development but becomes restricted to neurogenic regions in adulthood

• Co-localization with NSC markers:

  • In the adult hippocampal niche: BASP1 marks type I radial NSCs

  • In the SVZ: BASP1 is found in B and C cells

  • Co-stain with GFAP, Sox2, Nestin, and DCX to identify specific progenitor populations

• Translational applications:

  • Apply BASP1 antibodies to human brain organoids to study human neurogenesis

  • The NSC-6 antibody has been validated in human brain organoids

• Functional studies:

  • Isolate BASP1-positive cells by FACS for in vitro culture and differentiation assays

  • Assess the stemness properties of BASP1-high versus BASP1-low neural cells