LASP1 Antibody, Biotin conjugated

What is LASP1 and why is it significant in research?

LASP1 (LIM and SH3 domain protein 1) is a 261 amino acid protein that localizes to both the cytoplasm and cytoskeleton. It consists of an N-terminal LIM-domain with two zinc finger motifs, followed by two central actin-binding nebulin repeats, flanked by a linker region and a C-terminal SH3 domain . LASP1 interacts with F-Actin and plays an important role in the regulation of actin-associated cytoskeletal organization .

The protein is significantly overexpressed in several cancer entities including breast, ovarian, colon, prostate, liver, and bladder carcinoma as well as medulloblastoma . LASP1 expression and nuclear localization significantly correlate with poor outcome of cancer patients, making it an important research target in oncology . Additionally, LASP1 has been identified as part of a six-gene signature that strongly predicts disease progression and relapse in chronic myeloid leukemia (CML) patients .

What are the typical molecular characteristics of LASP1 in experimental detection?

When detecting LASP1 using antibody-based methods, researchers should note these characteristics:

The difference between calculated and observed molecular weights is due to post-translational modifications, particularly phosphorylation events that affect protein migration in SDS-PAGE .

How does biotin conjugation enhance LASP1 antibody utility in research applications?

Biotin conjugation offers several methodological advantages for LASP1 antibody applications:

• Enhanced sensitivity: The biotin-streptavidin system provides significant signal amplification due to the high affinity interaction (Kd~10^-15 M), enabling detection of low-abundance LASP1 in complex samples .

• Reduced background: In multi-step detection protocols, biotin conjugation eliminates species cross-reactivity issues that can occur with secondary antibodies.

• Versatile detection options: Researchers can use various streptavidin-conjugated detection reagents (HRP, fluorophores) with the same primary biotin-conjugated antibody.

• Compatibility with amplification systems: The biotin-conjugated antibody works effectively in tyramide signal amplification (TSA) systems for ultra-sensitive detection in tissues with low LASP1 expression .

• Multiplexing capability: When studying LASP1 interactions with proteins like CRKL, biotin-conjugated antibodies facilitate dual or triple labeling protocols without species constraints .

How can LASP1 Antibody, Biotin conjugated be used to study phosphorylation events in cancer research?

LASP1 phosphorylation, particularly at tyrosine-171 (Y171), is a critical regulatory event in cancer biology and can be effectively studied using biotin-conjugated antibodies with these methodological approaches:

• Dual detection strategies: Researchers can use biotin-conjugated LASP1 antibody alongside a phospho-specific antibody targeting pLASP1-Y171 to simultaneously assess total and phosphorylated protein levels . This approach reveals the proportion of LASP1 undergoing phosphorylation in response to experimental treatments.

• BCR-ABL signaling studies: In CML research, LASP1 has been identified as a direct substrate of BCR-ABL kinase, with specific phosphorylation at Y171. Biotin-conjugated LASP1 antibodies combined with tyrosine kinase inhibitor (TKI) treatments can monitor how drugs like nilotinib affect LASP1 phosphorylation status .

• Protein-protein interaction investigations: Phosphorylated LASP1 (pLASP1-Y171) binds to the SH2 domain of non-phosphorylated CRKL. Researchers can use biotin-conjugated LASP1 antibody in conjunction with proximity ligation assays to visualize these interactions in situ .

• Subcellular localization studies: While total LASP1 typically localizes at focal adhesions, lamellipodia, and membrane ruffles, phosphorylation at Y171 blocks LASP1 translocation to focal adhesions. Biotin-conjugated antibodies enable subcellular tracking of LASP1 populations through confocal microscopy .

• Temporal dynamics analysis: Sequential sampling and detection using biotin-conjugated LASP1 antibodies can reveal the kinetics of phosphorylation and dephosphorylation in response to stimuli, particularly in BCR-ABL positive cell lines like K562 and M07p210 .

What methodological approaches can resolve contradictory results in LASP1 detection between different experimental systems?

When researchers encounter contradictory results with LASP1 detection across experimental systems, these methodological approaches can help resolve discrepancies:

• Validation across multiple antibody epitopes: LASP1 antibodies target different regions (N-terminal AA 16-30, central region AA 128-252). Using multiple antibodies recognizing distinct epitopes can confirm specificity and rule out isoform-specific detection issues .

• Phosphorylation-dependent epitope masking: Consider whether phosphorylation at Y171 might mask antibody binding sites. Compare results with and without phosphatase treatment of samples to reveal potential epitope blocking .

• Cell line heterogeneity assessment: LASP1 expression and phosphorylation vary significantly between cell types. When comparing BCR-ABL positive (K562, M07p210) versus negative (M07e) cell lines, account for these differences in experimental design .

• Correlation with genetic manipulation: Validate antibody specificity through genetic approaches (siRNA knockdown, CRISPR knockout) coupled with antibody detection to establish true signal versus background .

• Sample preparation standardization: Differences in extraction buffers, fixation protocols, and blocking reagents can significantly impact LASP1 detection. Document and standardize these parameters across experiments .

• Technical considerations table:

How can researchers effectively use LASP1 Antibody, Biotin conjugated in multiplex immunoassays with other BCR-ABL pathway markers?

For effective multiplex analysis of BCR-ABL signaling using LASP1 Antibody, Biotin conjugated, researchers should implement these methodological approaches:

• Optimized sequential staining protocols: When combining LASP1 detection with other BCR-ABL pathway markers (CRKL, ABL, etc.), researchers should determine the optimal staining sequence. For fixed cell or tissue samples, begin with the least abundant target (often phospho-specific epitopes) and proceed to more abundant targets .

• Verified antibody compatibility: Before full multiplex experiments, verify that all antibodies in the panel perform consistently in both single-target and multiplex conditions. Test for potential cross-reactivity between detection systems .

• Strategic conjugate selection: When designing multiplex panels:

  • Use LASP1 Antibody, Biotin conjugated with streptavidin-fluorophore detection systems that have minimal spectral overlap with other fluorophores

  • Pair with directly conjugated antibodies for other targets

  • Consider tyramide signal amplification for low-abundance phosphorylated targets

• Validated controls for co-localization studies:

  • Positive controls: BCR-ABL positive cell lines (K562, M07p210) treated with/without nilotinib

  • Negative controls: BCR-ABL negative cell lines (M07e)

  • Single antibody controls to establish bleed-through parameters

• Quantification strategies: For co-localization analysis of LASP1 with binding partners like CRKL, implement:

  • Pearson's correlation coefficient measurement

  • Mander's overlap coefficient

  • Object-based co-localization for punctate structures

  • Quantitative co-immunoprecipitation as a complementary approach

• Example multiplex protocol for BCR-ABL signaling:

  a. Fix cells with 4% paraformaldehyde (10 minutes)
  b. Permeabilize with 0.1% saponin in PBS (30 minutes)
  c. Block with 3% donkey serum (30 minutes)
  d. Primary antibody incubation with phospho-specific antibodies
  e. Secondary detection with appropriate fluorophores
  f. Additional blocking step
  g. LASP1 Antibody, Biotin conjugated (1:200 dilution)
  h. Streptavidin-fluorophore detection
  i. Nuclear counterstain
  j. Confocal microscopy analysis

What are the optimal conditions for using LASP1 Antibody, Biotin conjugated in ELISA applications?

For optimal ELISA performance with LASP1 Antibody, Biotin conjugated, researchers should implement these protocol specifications:

• Coating conditions:

  • Antigen concentration: 1-10 μg/ml recombinant LASP1 in carbonate buffer (pH 9.6)

  • Incubation: Overnight at 4°C

  • Blocking: 3% BSA in PBS-T (PBS with 0.05% Tween-20) for 1 hour at room temperature

• Antibody dilution optimization:

  • Initial titration range: 1:200-1:2000

  • Determine optimal concentration using a standard curve of recombinant LASP1 protein

  • Include phosphorylated and non-phosphorylated LASP1 standards if studying phosphorylation status

• Detection system:

  • Streptavidin-HRP dilution: 1:1000-1:5000

  • Substrate: TMB solution for colorimetric detection

  • Stop solution: 2N H₂SO₄

  • Read absorbance at 450nm with 620nm reference

• Sample preparation recommendations:

  • Cell lysates: Prepare in RIPA buffer with phosphatase inhibitors

  • Protein concentration: 1-5 mg/ml total protein

  • Pre-clear lysates by centrifugation (14,000×g, 15 min, 4°C)

• Controls and validation:

  • Positive control: K562 cell lysate (BCR-ABL positive cell line)

  • Negative control: M07e cell lysate (BCR-ABL negative)

  • Background control: Detection system without primary antibody

  • Specificity control: LASP1-depleted lysate via immunoprecipitation

• Troubleshooting parameters:

What sample preparation protocols maximize LASP1 detection sensitivity while preserving phosphorylation status?

To maximize detection sensitivity while preserving LASP1 phosphorylation status, implement these critical methodological steps:

• Cell harvesting and initial processing:

  • Rapidly harvest cells by scraping in ice-cold PBS

  • Centrifuge at 200×g for 5 minutes at 4°C

  • Remove supernatant completely to avoid phosphatase activation

• Lysis buffer formulation:

  • Base buffer: 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40

  • Phosphatase inhibitors: 10 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA

  • Protease inhibitors: 1 mM PMSF, protease inhibitor cocktail

  • Reducing agent: 1 mM DTT (added fresh)

  • Buffer should be ice-cold before use

• Lysis procedure:

  • Resuspend cell pellet in lysis buffer (500 μl per 10⁷ cells)

  • Incubate on ice for 30 minutes with gentle vortexing every 5 minutes

  • Clarify lysate by centrifugation at 14,000×g for 15 minutes at 4°C

  • Transfer supernatant to new tube, avoid disturbing the pellet

• Subcellular fractionation considerations:

  • For compartment-specific analysis, use dedicated fractionation kits

  • Verify fractionation purity using compartment-specific markers:

    • Cytoplasmic: GAPDH

    • Nuclear: Lamin A/C

  • Analyze fractions separately, as phospho-LASP1 distribution varies between compartments

• Sample storage:

  • Aliquot lysates to avoid freeze-thaw cycles

  • Add glycerol to 10% final concentration

  • Flash-freeze in liquid nitrogen

  • Store at -80°C for no longer than 3 months

• Pre-analytical processing:

  • Determine protein concentration using Bradford or BCA assay

  • Normalize all samples to equal protein concentration

  • Add Laemmli sample buffer with phosphatase inhibitors

  • Heat at 95°C for 5 minutes for Western blotting applications

• Verification of phosphorylation preservation:

  • Include control phospho-proteins in your workflow (e.g., phospho-CRKL)

  • Compare phosphorylation levels with and without phosphatase inhibitors

  • Include samples with known phosphorylation status (e.g., nilotinib-treated versus untreated cells)

How should researchers design validation experiments to confirm LASP1 antibody specificity and performance reliability?

A comprehensive validation strategy for LASP1 antibody specificity and reliability should include these methodological approaches:

• Epitope verification:

  • Compare performance with antibodies targeting different LASP1 epitopes (N-terminal AA 16-30 vs. central region AA 128-252)

  • Verify recognition patterns across species according to epitope conservation (human, mouse, rat)

  • Use pre-adsorption tests with immunizing peptide to confirm specific binding

• Genetic validation controls:

  • siRNA knockdown: Transfect cells with LASP1-specific siRNA and control siRNA

  • CRISPR/Cas9 knockout: Generate LASP1 knockout cell lines

  • Overexpression: Compare wild-type cells with LASP1-overexpressing models

  • Document antibody signal reduction proportional to protein reduction

• Cross-platform verification protocol:

  • Western blot: Confirm single band at expected MW (35-38 kDa)

  • Immunoprecipitation: Pull down LASP1 and verify by mass spectrometry

  • Immunofluorescence: Compare patterns with published subcellular localization

  • Flow cytometry: Assess correlation between signal and known expression levels

• Phosphorylation-specific validation:

  • Compare antibody performance in paired samples:

    • BCR-ABL-positive (K562) vs. BCR-ABL-negative (M07e) cells

    • Nilotinib-treated vs. untreated cells

    • Time course after inhibitor treatment (30 min, 24h, 48h)

  • Verify phosphorylation status with phospho-specific antibodies

• Reproducibility assessment matrix:

• Extended validation for biotin conjugation:

  • Compare conjugated vs. unconjugated antibody performance

  • Test with different streptavidin detection systems (HRP, fluorophores)

  • Assess signal-to-noise ratio across detection platforms

  • Determine optimal streptavidin concentration for each application

How can researchers address non-specific binding and high background issues when working with LASP1 Antibody, Biotin conjugated?

When encountering non-specific binding or high background with LASP1 Antibody, Biotin conjugated, implement these evidence-based troubleshooting approaches:

• Blocking optimization strategy:

  • Test multiple blocking agents: 5% BSA, 5% milk, commercial blocking buffers

  • Note: For phosphorylation studies, avoid milk as it contains phospho-proteins

  • Extend blocking time to 2 hours at room temperature

  • Add 0.1-0.3% Triton X-100 to blocking buffer for improved penetration

• Endogenous biotin management:

  • Pre-block endogenous biotin using an avidin/biotin blocking kit

  • For tissues with high endogenous biotin (liver, kidney), implement sequential avidin and biotin blocking steps

  • Consider using fluorescently-labeled streptavidin rather than enzyme-conjugated for lower background

• Streptavidin detection optimization:

  • Titrate streptavidin concentration (1:500 to 1:5000)

  • Include 0.1% Tween-20 in wash and incubation buffers

  • For HRP-streptavidin, limit substrate development time and monitor closely

  • For fluorescent detection, include an extra wash step with high-salt buffer (500 mM NaCl)

• Antibody dilution and incubation adjustment:

  • Increase antibody dilution (start with 1:1000 and test up to 1:5000)

  • Reduce incubation temperature (4°C overnight instead of room temperature)

  • Add 0.1% BSA to antibody dilution buffer to reduce non-specific interactions

• Sample-specific considerations:

  • For fixed tissues: Quench autofluorescence with 0.1% Sudan Black B

  • For cell lines: Verify fixation protocol compatibility (methanol vs. paraformaldehyde)

  • For lysates: Pre-clear with Protein G beads before immunoprecipitation

  • For patient samples: Include additional blocking with 10% serum from the host species

• Systematic background reduction protocol:

What are the critical factors affecting LASP1 phosphorylation detection in experimental systems?

For reliable detection of LASP1 phosphorylation states in experimental systems, researchers should address these critical factors:

• Phosphatase activity management:

  • Include multiple phosphatase inhibitors (NaF, Na₃VO₄, β-glycerophosphate)

  • Maintain samples at 4°C throughout processing

  • Minimize time between collection and analysis/preservation

  • Use dedicated phospho-preservation lysis buffers

• BCR-ABL activity considerations:

  • Document BCR-ABL expression/activity levels in experimental systems

  • For inhibitor studies, verify BCR-ABL inhibition using established markers (phospho-CRKL)

  • Account for cell-specific differences in kinase/phosphatase balance

  • Include control cell lines with defined BCR-ABL status (K562, M07p210, M07e)

• Epitope accessibility factors:

  • Phosphorylation at Y171 may mask nearby epitopes

  • For total LASP1 detection, select antibodies targeting regions distant from Y171

  • For dual detection of total and phospho-LASP1, use antibodies raised in different species

  • Consider native vs. denatured detection limitations

• Temporal dynamics awareness:

  • LASP1 phosphorylation changes rapidly after BCR-ABL inhibition

  • Document precise timing of treatments and sample collection

  • For kinetic studies, include multiple timepoints (30 min, 2h, 24h, 48h)

  • Consider pulse-chase experiments for turnover assessment

• Detection method selection matrix:

• Standardization recommendations:

  • Include both positive controls (BCR-ABL+ cells) and negative controls (TKI-treated cells)

  • Document percentage of LASP1 phosphorylation relative to total LASP1

  • Use recombinant phospho-LASP1 standards for absolute quantification

  • Validate phospho-specific antibodies with phosphatase-treated samples

How can researchers effectively compare data across different LASP1 antibody-based detection systems in collaborative projects?

To ensure reliable cross-comparison of LASP1 data between laboratories using different antibody detection systems, implement these standardization practices:

• Reference standard implementation:

  • Establish common control samples distributed to all participating labs

  • Include recombinant LASP1 standard curves at defined concentrations

  • Share positive control lysates (K562 cells) prepared in bulk and aliquoted

  • Create a phosphorylation standard using in vitro kinase reactions with recombinant LASP1

• Antibody characterization exchange:

  • Document complete antibody information:

    • Clone/catalog number

    • Epitope region

    • Host species

    • Conjugation type

    • Validation data

  • Cross-validate antibodies against the same sample set before main experiments

• Protocol harmonization strategy:

  • Develop detailed shared protocols with specific reagents and suppliers

  • Control critical variables (lysis buffer composition, antibody dilution, incubation times)

  • Use consistent imaging/detection parameters across sites

  • Include protocol adherence verification steps

• Quantification standardization:

  • Adopt common quantification methods and software

  • Use relative quantification with agreed-upon housekeeping controls

  • Implement normalization to total protein (REVERT total protein stain or similar)

  • Establish acceptance criteria for inter-lab coefficient of variation (<20%)

• Technical replicate design:

  • Each lab processes duplicate or triplicate samples

  • Include site-to-site sample exchange for direct comparison

  • Perform analysis both within and between laboratories

  • Document and address systematic biases

• Data integration framework:

• Cross-validation with orthogonal techniques:

  • Confirm key findings using alternative detection methods

  • Supplement antibody-based detection with mass spectrometry

  • Validate protein-protein interactions with multiple techniques (co-IP, PLA, FRET)

  • Use genetic approaches to confirm biological significance of findings

By implementing these standardized approaches, research teams can effectively combine data from different laboratories using various LASP1 antibody detection systems while maintaining scientific rigor and reproducibility.