SFR1 Antibody, Biotin conjugated

What is SFR1 and why is it targeted in research applications?

SFR1 (SWI5-Dependent Recombination Repair 1) is a protein involved in DNA repair mechanisms, particularly in homologous recombination pathways. As a component of the DNA repair machinery, SFR1 plays a critical role in maintaining genomic stability. Antibodies targeting SFR1 are valuable tools for investigating DNA damage response mechanisms, recombination processes, and associated cellular pathways. The anti-SFR1 antibody is typically generated from rabbits immunized with a KLH conjugated synthetic peptide corresponding to amino acids 1-30 from the N-terminal region of human SFR1 (previously known as C10orf78) .

What is the significance of biotin conjugation for SFR1 antibodies?

Biotin conjugation of SFR1 antibodies leverages the biotin-(strept)avidin system, which features one of the strongest non-covalent interactions in nature, approximately 10³ to 10⁶ times higher affinity than typical antigen-antibody interactions . This conjugation strategy provides significant advantages for detection sensitivity, including signal amplification capabilities that enhance detection of low-abundance SFR1 protein. The biotin tag allows for versatile detection methods through secondary streptavidin-conjugated reporters (enzymes, fluorophores) without altering the antibody's binding specificity to the SFR1 target .

How does the biotin-(strept)avidin interaction enhance SFR1 detection compared to direct detection methods?

The biotin-(strept)avidin system offers substantial technical advantages for SFR1 detection over direct methods. The interaction's extraordinary stability against manipulation, proteolytic enzymes, temperature and pH extremes, and harsh organic reagents makes it exceptionally reliable for experimental protocols . In practical applications, this interaction enables:

• Signal amplification: Each biotin-conjugated SFR1 antibody can bind multiple streptavidin molecules, each carrying multiple reporter molecules

• Enhanced sensitivity: Detection of very low concentrations of SFR1 protein becomes feasible

• Reduced background: The specificity of the interaction minimizes non-specific signals

• Flexible detection options: Compatible with various streptavidin-conjugated reporters (enzymes, fluorophores, quantum dots)

Which experimental techniques are compatible with biotin-conjugated SFR1 antibodies?

Biotin-conjugated SFR1 antibodies demonstrate versatility across multiple experimental platforms. Based on established protocols for similar biotin-conjugated antibodies, the SFR1 antibody (AA 1-30) with biotin conjugation is validated for:

• Western Blotting (WB): Detecting SFR1 protein in cell/tissue lysates with streptavidin-HRP secondary detection

• Enzyme-Linked Immunosorbent Assay (ELISA): Quantitative measurement of SFR1 protein levels

• Flow Cytometry (FACS): Analysis of cellular SFR1 expression in single-cell suspensions

Additional applications supported by the biotin-conjugation platform may include immunohistochemistry, immunoprecipitation, and chromatin immunoprecipitation, though specific validation for SFR1 antibodies in these applications should be confirmed.

What is the optimal protocol for using biotin-conjugated SFR1 antibodies in Western blotting experiments?

For optimal Western blotting results with biotin-conjugated SFR1 antibodies:

• Sample preparation:

  • Lyse cells in RIPA buffer containing protease inhibitors

  • Determine protein concentration (BCA or Bradford assay)

  • Denature 20-40 μg protein with Laemmli buffer (95°C, 5 minutes)

• Electrophoresis and transfer:

  • Separate proteins on 10-12% SDS-PAGE gel

  • Transfer to PVDF or nitrocellulose membrane (wet transfer recommended)

• Immunodetection:

  • Block membrane with 5% non-fat milk or BSA in TBST (1 hour, room temperature)

  • Incubate with biotin-conjugated SFR1 antibody (1:300-1:5000 dilution) overnight at 4°C

  • Wash 3x with TBST (10 minutes each)

  • Incubate with streptavidin-HRP (1:10,000-1:20,000) for 1 hour at room temperature

  • Wash 3x with TBST (10 minutes each)

  • Develop using ECL substrate and image

• Controls:

  • Include positive control (cell line with known SFR1 expression)

  • Include loading control (β-actin, GAPDH)

  • Consider including a blocking peptide control to verify specificity

How can biotin-conjugated SFR1 antibodies be optimized for flow cytometry applications?

For flow cytometry applications with biotin-conjugated SFR1 antibodies:

• Cell preparation:

  • Harvest cells (1-5 × 10⁶ cells per sample)

  • Fix with 4% paraformaldehyde (10 minutes, room temperature)

  • For intracellular staining, permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer

• Staining protocol:

  • Block with 1-5% BSA in PBS (30 minutes, room temperature)

  • Incubate with biotin-conjugated SFR1 antibody at optimized dilution (typically 1:100-1:500) for 30-60 minutes at 4°C

  • Wash 2-3× with PBS containing 1% BSA

  • Incubate with streptavidin-fluorophore conjugate (PE, APC, FITC) at manufacturer's recommended dilution for 30 minutes at 4°C in the dark

  • Wash 2-3× with PBS containing 1% BSA

  • Resuspend in appropriate buffer for analysis

• Optimization steps:

  • Titrate antibody concentration to determine optimal signal-to-noise ratio

  • Include appropriate compensation controls when using multiple fluorophores

  • Incorporate isotype controls to assess non-specific binding

  • Consider using viability dye to exclude dead cells from analysis

What are the common issues when using biotin-conjugated SFR1 antibodies and how can they be resolved?

How can endogenous biotin interference be minimized when using biotin-conjugated SFR1 antibodies?

Endogenous biotin can interfere with detection systems that rely on the biotin-streptavidin interaction, particularly in biotin-rich tissues like liver, kidney, and brain. To minimize this interference:

• Implement avidin/biotin blocking:

  • Prior to primary antibody incubation, block with unconjugated avidin (10-30 minutes)

  • Rinse briefly

  • Block with unconjugated biotin (10-30 minutes)

  • Rinse thoroughly before proceeding with primary antibody incubation

• Alternative fixation methods:

  • Use methanol or acetone fixation instead of formaldehyde when possible

  • Reduce fixation time to minimize biotin exposure

• Sample preparation considerations:

  • For tissue sections, thinner sections (3-5 μm) may reduce endogenous biotin exposure

  • For cell cultures, serum starvation prior to fixation may reduce biotin levels

• Detection alternatives:

  • Consider tyramide signal amplification as an alternative to direct streptavidin detection

  • In extreme cases of interference, consider using non-biotin conjugated antibodies with direct detection systems

What storage conditions maximize the shelf-life of biotin-conjugated SFR1 antibodies?

To maintain optimal activity of biotin-conjugated SFR1 antibodies:

• Recommended storage conditions:

  • Store at -20°C in a non-frost-free freezer

  • Keep in aqueous buffered solution containing 0.01M TBS (pH 7.4) with 1% BSA, 0.03% Proclin300, and 50% Glycerol

  • Expected stability: 12 months under proper storage conditions

• Handling practices:

  • Divide into small single-use aliquots upon receipt to minimize freeze-thaw cycles

  • Thaw aliquots at room temperature and briefly centrifuge before opening

  • Keep on ice when in use during experiments

  • Avoid repeated freeze-thaw cycles (limit to <5 cycles)

• Working solution stability:

  • Diluted antibody is typically stable for up to 7 days at 4°C

  • For longer storage of working dilutions, add carrier protein (0.1-1% BSA)

  • Monitor for precipitation or changes in color which may indicate degradation

How can biotin-conjugated SFR1 antibodies be utilized in multi-parameter imaging experiments?

Multi-parameter imaging with biotin-conjugated SFR1 antibodies enables simultaneous visualization of SFR1 alongside other cellular components. This approach provides valuable spatial information about SFR1's relationship with interaction partners or subcellular structures. Advanced implementation strategies include:

• Sequential multiplexing protocol:

  • Perform standard immunostaining with biotin-conjugated SFR1 antibody

  • Detect with streptavidin-conjugated fluorophore (e.g., streptavidin-Alexa Fluor 488)

  • Capture images

  • Elute or quench first-round antibodies

  • Repeat with additional markers using distinct fluorophores

  • Combine images for co-localization analysis

• Simultaneous multiplexing considerations:

  • Combine biotin-conjugated SFR1 antibody with directly-labeled antibodies of different species

  • Carefully select fluorophores to minimize spectral overlap

  • Implement appropriate controls to assess bleed-through

  • Consider spectral unmixing algorithms for closely overlapping fluorophores

• Super-resolution microscopy applications:

  • Use streptavidin conjugated to photoconvertible fluorophores for STORM/PALM

  • Employ streptavidin-quantum dots for enhanced photostability in extended imaging

  • Optimize antibody concentration to achieve appropriate labeling density

What experimental design considerations are important when studying SFR1 protein interactions using biotin-conjugated antibodies?

When investigating SFR1 protein interactions:

• Co-immunoprecipitation strategy:

  • Use biotin-conjugated SFR1 antibody to capture SFR1 and associated proteins

  • Precipitate complexes with streptavidin beads

  • Elute and analyze interacting partners by mass spectrometry or western blotting

  • Consider epitope accessibility: the SFR1 antibody (AA 1-30) targets the N-terminal region, which may be involved in certain protein interactions

• Proximity ligation assay (PLA) approach:

  • Combine biotin-conjugated SFR1 antibody with antibody against potential interaction partner

  • Use streptavidin-oligonucleotide and secondary antibody-oligonucleotide probes

  • Implement rolling circle amplification for detection of close proximity (<40 nm)

  • Quantify interaction events at the single-molecule level

• FRET/BRET considerations:

  • Pair biotin-conjugated SFR1 antibody with streptavidin-fluorophore (donor)

  • Label potential interaction partner with acceptor fluorophore

  • Measure energy transfer as indicator of molecular proximity

  • Control for potential steric effects of antibody-biotin-streptavidin complex

How can biotin-conjugated SFR1 antibodies be integrated into chromatin immunoprecipitation (ChIP) protocols?

Adapting ChIP protocols for biotin-conjugated SFR1 antibodies:

• Modified ChIP protocol:

  • Crosslink cells with 1% formaldehyde (10 minutes, room temperature)

  • Lyse cells and sonicate chromatin to 200-500 bp fragments

  • Pre-clear chromatin with protein A/G beads

  • Incubate chromatin with biotin-conjugated SFR1 antibody overnight at 4°C

  • Add streptavidin magnetic beads for 2-4 hours at 4°C

  • Wash stringently (low salt, high salt, LiCl, TE buffers)

  • Reverse crosslinks and purify DNA

  • Analyze by qPCR or sequencing

• Technical considerations:

  • The large streptavidin-biotin complex may impact efficiency; optimize antibody:chromatin ratios

  • Consider using biotinylated protein A/G instead of direct biotin conjugation for improved orientation

  • Include controls for non-specific binding of biotin to endogenous biotinylated proteins

  • Implement stringent washing steps to reduce background

• Sequential ChIP (Re-ChIP) application:

  • First round: Conventional ChIP with non-biotinylated antibody against chromatin modifier

  • Elute complexes under mild conditions

  • Second round: ChIP with biotin-conjugated SFR1 antibody

  • This approach identifies genomic regions where SFR1 and specific chromatin features co-occur

What are the relative advantages and limitations of biotin-conjugated versus directly labeled SFR1 antibodies?

How do different immunoassay platforms impact the performance of biotin-conjugated SFR1 antibodies?

Different immunoassay platforms present unique considerations for biotin-conjugated SFR1 antibody applications:

• ELISA considerations:

  • Conventional sandwich ELISA: Biotin-conjugated SFR1 antibody can serve as detection antibody with streptavidin-HRP for signal development

  • Bridged Avidin-Biotin (BRAB) method: Enhances sensitivity through additional signal amplification steps

  • Competitive ELISA: May use biotin-conjugated SFR1 antigen competing with sample SFR1 for antibody binding

• Flow cytometry platform effects:

  • Increased sensitivity compared to direct conjugates due to signal amplification

  • Additional washing steps required, potentially increasing cell loss

  • Multiple fluorophore options through different streptavidin conjugates

• Proximity-based assays (AlphaLISA, HTRF):

  • Biotin-conjugated SFR1 antibody paired with streptavidin-donor beads

  • Second antibody conjugated to acceptor beads

  • Energy transfer occurs only when antibodies bind same SFR1 molecule

  • Provides homogeneous (no-wash) detection format

• Imaging applications:

  • Immunohistochemistry: Biotin-conjugated antibodies may show higher sensitivity but require biotin blocking steps

  • High-content screening: Signal amplification beneficial for detecting low-abundance SFR1

  • Super-resolution microscopy: Additional streptavidin layer may impact spatial resolution

What methodological advances integrate biotin-conjugated antibodies with emerging single-cell technologies?

Recent technological integrations of biotin-conjugated antibodies with single-cell platforms offer new opportunities for SFR1 research:

• Mass cytometry (CyTOF) applications:

  • Biotin-conjugated SFR1 antibody followed by metal-tagged streptavidin

  • Enables integration into high-parameter panels (40+ markers)

  • Eliminates fluorescence spillover concerns

  • Protocol modification: longer incubation times and additional washing steps

• Single-cell sequencing integration:

  • CITE-seq approach: Biotin-conjugated SFR1 antibody with streptavidin-oligonucleotide tags

  • Simultaneously profiles SFR1 protein expression and transcriptome

  • Allows correlation between SFR1 protein levels and gene expression patterns

  • Technical consideration: optimize concentration to avoid barcode swapping

• Spatial transcriptomics combination:

  • Biotin-conjugated SFR1 antibody with streptavidin-fluorophore detection

  • Combine with in situ hybridization for spatial gene expression

  • Provides tissue context for SFR1 protein localization and associated gene expression

  • Implementation requires careful optimization of multiplexed protocols

• Microfluidic protein analysis:

  • Immobilize streptavidin in microchannels to capture biotin-conjugated SFR1 antibody complexes

  • Enables single-cell protein secretion analysis

  • Can be combined with other cellular readouts for multiparameter analysis

  • Requires optimization of surface chemistry and flow rates