SPAC4G9.14 Antibody

What applications has the SPAC4G9.14 Antibody been validated for?

The SPAC4G9.14 Antibody (product code CSB-PA608762XA01SXV) has been validated for several common research applications:

When designing experiments, researchers should perform preliminary validation studies to determine optimal conditions for their specific experimental system. Antibody validation typically includes positive controls (wild-type S. pombe) and negative controls (deletion strains lacking the target protein) .

How should SPAC4G9.14 Antibody be stored and handled to maintain optimal activity?

Proper storage and handling are critical for maintaining antibody functionality:

• Upon receipt, store the antibody at -20°C or -80°C according to manufacturer recommendations .

• Avoid repeated freeze-thaw cycles by aliquoting the antibody into single-use volumes before freezing.

• Store in a buffer containing 50% glycerol, 0.01M PBS, pH 7.4, and 0.03% Proclin 300 as a preservative .

• For short-term use (within two weeks), antibody can be stored at 4°C.

• Prior to use, thaw aliquots completely and gently mix—avoid vortexing, which can denature antibody proteins.

• Working dilutions should be prepared just before use and stored at 4°C for no more than 24 hours.

This storage protocol helps preserve epitope recognition capacity and prevents bacterial contamination that could compromise experimental results.

What controls should be included when working with SPAC4G9.14 Antibody?

Proper experimental controls are essential for antibody-based experiments:

• Positive control: Wild-type S. pombe strain 972 (ATCC 24843) expressing the endogenous SPAC4G9.14 protein .

• Negative control:

  • Primary antibody omission control (all reagents except primary antibody)

  • Ideally, a SPAC4G9.14 deletion strain if available

  • Non-expressing cells or tissues as appropriate

• Loading control: For Western blots, use antibodies against constitutively expressed proteins like actin or tubulin in S. pombe.

• Isotype control: A non-specific rabbit IgG at the same concentration as the primary antibody to identify potential non-specific binding.

Incorporating these controls enables accurate interpretation of results by distinguishing specific signals from background or non-specific interactions.

How can I optimize Western blot protocols specifically for SPAC4G9.14 Antibody?

Optimizing Western blot protocols for SPAC4G9.14 Antibody requires systematic adjustment of several parameters:

• Sample preparation:

  • Lyse S. pombe cells with glass beads in lysis buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.0), 0.5% Triton X-100, and 0.5% deoxycholate .

  • Include protease inhibitors: 0.4 mM phenylmethylsulfonyl fluoride and 1× protease inhibitor cocktail .

  • Heat samples at 95°C for 5 minutes in Laemmli buffer containing β-mercaptoethanol.

• Gel electrophoresis and transfer:

  • Use 15% polyacrylamide gels for optimal resolution of the target protein .

  • Transfer to nitrocellulose membranes at 100V for 1 hour or 30V overnight at 4°C.

• Blocking and antibody incubation:

  • Block membrane with 5% non-fat dry milk in TBST for 1 hour at room temperature.

  • Incubate with SPAC4G9.14 Antibody (1:1000 dilution) overnight at 4°C.

  • Wash 3× with TBST, 10 minutes each.

  • Incubate with HRP-conjugated secondary antibody (1:5000) for 1 hour at room temperature.

  • Wash 3× with TBST, 10 minutes each.

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) with exposure times ranging from 30 seconds to 5 minutes.

  • For weak signals, consider using ECL Prime or other high-sensitivity substrates.

Troubleshooting tip: If background is high, increase washing times and consider using 1% BSA instead of milk for blocking.

What are recommended protocols for immunoprecipitation using SPAC4G9.14 Antibody?

An optimized immunoprecipitation protocol for SPAC4G9.14 Antibody requires careful attention to buffer components and washing conditions:

• Cell lysis and extract preparation:

  • Harvest 50-100 ml of S. pombe culture (OD600 = 0.5-1.0).

  • Wash cells with cold PBS and resuspend in IP lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate).

  • Add protease inhibitors (1 mM PMSF, 1 μg/ml leupeptin, 1 μg/ml pepstatin).

  • Lyse cells with glass beads using a bead beater (4 cycles of 30 seconds with 30-second intervals on ice).

  • Clear lysate by centrifugation at 12,000 × g for 10 minutes at 4°C.

• Pre-clearing and immunoprecipitation:

  • Pre-clear lysate with Protein A/G beads for 1 hour at 4°C.

  • Incubate pre-cleared lysate with 2-5 μg SPAC4G9.14 Antibody overnight at 4°C with gentle rotation.

  • Add 50 μl Protein A/G beads and incubate for 2-3 hours at 4°C.

  • Collect beads by centrifugation and wash 4× with IP wash buffer (same as lysis buffer but with 0.1% Triton X-100).

• Elution and analysis:

  • Elute bound proteins by boiling in 2× SDS sample buffer for 5 minutes.

  • Analyze by SDS-PAGE and Western blotting.

For confirmation of specificity, perform parallel IPs with non-specific rabbit IgG and compare the band patterns.

How can I troubleshoot weak or absent signals when using SPAC4G9.14 Antibody?

When encountering weak or absent signals with SPAC4G9.14 Antibody, systematically address these potential issues:

• Antibody functionality:

  • Verify antibody activity with a dot blot using purified recombinant protein.

  • Check antibody expiration date and storage conditions.

  • Consider testing a new lot or aliquot.

• Sample preparation issues:

  • Ensure complete cell lysis by examining cell debris microscopically.

  • Verify protein extraction by measuring total protein concentration.

  • Confirm protein transfer efficiency with reversible staining (Ponceau S).

  • Increase protein concentration loaded (up to 50-100 μg/lane).

• Protocol optimization:

  • Reduce antibody dilution (use more concentrated antibody).

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

  • Use alternative detection methods (e.g., switch from ECL to fluorescent detection).

  • Try different blocking agents (BSA vs. milk vs. commercial blockers).

• Expression considerations:

  • Verify target protein expression under your experimental conditions.

  • Consider inducing or upregulating expression if possible.

  • Check if protein expression is cell cycle-dependent or condition-specific.

Systematic troubleshooting table:

What are the recommended conditions for immunofluorescence using SPAC4G9.14 Antibody?

For optimal immunofluorescence with SPAC4G9.14 Antibody in S. pombe, follow this detailed protocol:

• Cell preparation and fixation:

  • Grow S. pombe to mid-log phase (OD600 = 0.5-0.7).

  • Fix cells with 3.7% formaldehyde for 30 minutes at room temperature.

  • Wash 3× with PEM buffer (100 mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgSO4).

  • Digest cell wall with Zymolyase 20T (1 mg/ml in PEM with 1.2 M sorbitol) for 30-60 minutes at 37°C.

  • Permeabilize with 1% Triton X-100 in PEM for 5 minutes.

• Blocking and antibody incubation:

  • Block with PEMBAL (PEM + 1% BSA, 0.1% sodium azide, 100 mM lysine hydrochloride) for 30 minutes.

  • Incubate with SPAC4G9.14 Antibody diluted 1:100 in PEMBAL overnight at 4°C.

  • Wash 3× with PEMBAL, 10 minutes each.

  • Incubate with fluorescent secondary antibody (Alexa Fluor 488 or similar) diluted 1:500 in PEMBAL for 2 hours at room temperature.

  • Wash 3× with PEMBAL, 10 minutes each.

• Nuclear staining and mounting:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes.

  • Mount slides with anti-fade mounting medium.

• Imaging parameters:

  • Use confocal microscopy with appropriate filter sets.

  • Acquire Z-stacks with 0.3-0.5 μm spacing.

  • Use identical exposure settings for all samples and controls.

This protocol should yield specific staining patterns that can be correlated with the subcellular localization of the SPAC4G9.14 protein.

How do I design experiments to validate SPAC4G9.14 Antibody specificity?

Validating antibody specificity is critical for ensuring reliable research results. For SPAC4G9.14 Antibody, implement these validation approaches:

• Genetic validation:

  • Compare antibody signal between wild-type S. pombe and a SPAC4G9.14 deletion mutant.

  • Use CRISPR-Cas9 to tag the endogenous protein and compare antibody staining with tag-specific antibodies.

  • Perform siRNA/shRNA knockdown and observe corresponding reduction in antibody signal.

• Biochemical validation:

  • Perform peptide competition assays using the immunizing peptide to block antibody binding.

  • Conduct Western blots with recombinant protein containing partial or complete sequence of SPAC4G9.14.

  • Use mass spectrometry to identify proteins in immunoprecipitates obtained with the antibody.

• Orthogonal validation:

  • Compare results with an independent antibody targeting a different epitope of SPAC4G9.14.

  • Correlate protein expression detected by the antibody with mRNA levels measured by RT-qPCR.

  • For tagged proteins, compare antibody detection with direct tag detection.

• Cross-reactivity assessment:

  • Test the antibody against related proteins or in other yeast species.

  • Perform Western blots on whole cell lysates to identify additional bands that may represent cross-reactivity.

Document all validation experiments thoroughly according to antibody validation guidelines to ensure reproducibility and reliability of subsequent experiments.

What are appropriate positive and negative controls for SPAC4G9.14 expression studies?

Establishing proper controls for SPAC4G9.14 expression studies ensures reliable data interpretation:

Positive controls:

• Wild-type S. pombe strain 972 (ATCC 24843) with confirmed SPAC4G9.14 expression .

• Cells under conditions known to induce SPAC4G9.14 expression (if characterized).

• Recombinant SPAC4G9.14 protein (for Western blot standard curve).

• SPAC4G9.14 overexpression strain using an inducible promoter system.

Negative controls:

• SPAC4G9.14 deletion strain (if available).

• SPAC4G9.14 knockdown using RNAi or other gene silencing approaches.

• Cells from growth conditions that downregulate SPAC4G9.14 expression (if known).

• Non-expressing organisms (e.g., different yeast species lacking close SPAC4G9.14 homologs).

Procedural controls:

• Primary antibody omission control to assess secondary antibody specificity.

• Isotype control (non-specific rabbit IgG) to evaluate non-specific binding.

• Peptide competition control to confirm epitope specificity.

• Loading controls (e.g., anti-actin antibody) to normalize for total protein amount.

Implement these controls systematically across all experiments to ensure data reliability and facilitate accurate interpretation of SPAC4G9.14 expression patterns.

How can I quantify SPAC4G9.14 protein expression levels accurately?

Accurate quantification of SPAC4G9.14 protein expression requires appropriate methodology and careful control implementation:

• Western blot quantification:

  • Use a dilution series of recombinant SPAC4G9.14 protein to create a standard curve.

  • Apply equal amounts of total protein (20-50 μg) for each sample.

  • Include reference proteins (actin, tubulin) as loading controls.

  • Use fluorescent secondary antibodies for wider linear dynamic range compared to chemiluminescence.

  • Capture images using a digital imaging system (e.g., LI-COR Odyssey, Bio-Rad ChemiDoc).

  • Perform densitometry using ImageJ or similar software, normalizing to loading controls.

• Flow cytometry quantification:

  • Fix and permeabilize cells using optimized protocols for yeast.

  • Stain with SPAC4G9.14 Antibody followed by fluorophore-conjugated secondary antibody.

  • Include unstained, secondary-only, and isotype controls.

  • Use quantitative beads with known antibody binding capacity to establish a standard curve.

  • Calculate molecules of equivalent soluble fluorochrome (MESF) to estimate protein abundance.

• Mass spectrometry-based quantification:

  • Use stable isotope-labeled peptides corresponding to unique SPAC4G9.14 sequences as internal standards.

  • Perform selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) for targeted quantification.

  • Calculate absolute protein amounts using the ratio of endogenous to labeled peptide signals.

• ELISA-based quantification:

  • Develop a sandwich ELISA using SPAC4G9.14 Antibody and a second antibody recognizing a different epitope.

  • Create a standard curve using purified recombinant protein.

  • Ensure sample dilutions fall within the linear range of the assay.

For all methods, biological replicates (n≥3) and technical replicates are essential for statistical validity.

What experimental approaches are suitable for studying SPAC4G9.14 protein-protein interactions?

Several complementary approaches can be used to investigate SPAC4G9.14 protein-protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use SPAC4G9.14 Antibody to pull down the protein complex from S. pombe lysates.

  • Identify interacting partners by Western blot (for known candidates) or mass spectrometry (for discovery).

  • Validate interactions by reverse Co-IP using antibodies against identified partners.

  • Protocol: Use gentle lysis conditions (150 mM NaCl, 0.5% NP-40) to preserve protein complexes .

• Proximity-based labeling:

  • Generate fusion proteins of SPAC4G9.14 with BioID, TurboID, or APEX2.

  • Express in S. pombe to biotinylate proximal proteins.

  • Purify biotinylated proteins using streptavidin beads and identify by mass spectrometry.

  • This approach captures transient interactions and works in native cellular environments.

• Yeast two-hybrid (Y2H) screening:

  • Use SPAC4G9.14 as bait in conventional Y2H or split-ubiquitin Y2H systems.

  • Screen against S. pombe cDNA library or arrays of known proteins.

  • Validate positive interactions with secondary assays like Co-IP.

• Förster Resonance Energy Transfer (FRET):

  • Create fluorescent protein fusions (e.g., SPAC4G9.14-CFP and candidate partner-YFP).

  • Measure FRET efficiency using acceptor photobleaching or fluorescence lifetime imaging.

  • Provides spatial information about interactions in living cells.

• Cross-linking mass spectrometry (XL-MS):

  • Treat living S. pombe cells with membrane-permeable crosslinkers.

  • Purify SPAC4G9.14 complexes under denaturing conditions.

  • Identify crosslinked peptides by mass spectrometry to map interaction interfaces.

Interaction data should be organized in a network diagram with confidence scores based on detection across multiple methods.

How do I account for background signal when analyzing SPAC4G9.14 Antibody data?

Properly addressing background signal is essential for accurate data interpretation:

• Background identification and correction strategies:

  • For Western blots: Subtract lane-specific background from regions adjacent to bands of interest.

  • For immunofluorescence: Measure and subtract background from cell-free regions within the same field.

  • For flow cytometry: Set gates based on negative controls (unstained cells and isotype controls).

  • For ELISA: Subtract values from blank wells containing all reagents except primary antibody.

• Control-based normalization:

  • Measure signal in negative control samples (knockout/knockdown cells).

  • Calculate signal-to-noise ratio by dividing specific signal by background signal.

  • Consider signal valid only when signal-to-noise ratio exceeds 3:1.

• Image analysis approaches:

  • Use software like ImageJ with rolling ball background subtraction.

  • Implement multi-parameter thresholding to distinguish specific signal from background.

  • Apply deconvolution algorithms for fluorescence microscopy data.

• Statistical approaches:

  • Use Receiver Operating Characteristic (ROC) curves to determine optimal thresholds.

  • Apply machine learning algorithms to separate signal from background in complex datasets.

  • Conduct bootstrapping to estimate confidence intervals around true signal measurements.

For all quantitative analyses, document background correction methods thoroughly to ensure reproducibility.

How can I distinguish between specific and non-specific signals in SPAC4G9.14 Antibody experiments?

Distinguishing specific from non-specific signals requires systematic controls and analytical approaches:

• Control-based validation:

  • Genetic controls: Compare signals between wild-type and SPAC4G9.14 knockout cells.

  • Antibody controls: Compare with isotype control antibody at equivalent concentration.

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

  • Secondary-only control: Omit primary antibody to detect non-specific secondary binding.

• Signal characteristics analysis:

  • Molecular weight: Specific bands should appear at the predicted molecular weight.

  • Subcellular localization: Compare observed localization with predicted patterns.

  • Response to treatments: Specific signals should change predictably with treatments affecting the target.

  • Consistency: Specific signals should be consistent across different detection methods.

• Quantitative approaches:

  • Calculate signal-to-noise ratio and set minimum threshold (typically >3:1).

  • Perform dose-response experiments with antibody dilutions.

  • Use dual-labeling with another antibody or tag-based detection system.

• Pattern recognition strategies:

  • In immunofluorescence, compare with known markers of subcellular compartments.

  • Identify common patterns of non-specific binding (e.g., nuclear envelope, cell wall).

  • Use image analysis software to create masks based on control images.

Systematic approach to signal validation:

How can SPAC4G9.14 Antibody be optimized for chromatin immunoprecipitation (ChIP) experiments?

Optimizing ChIP protocols for SPAC4G9.14 Antibody requires special considerations for yeast cells:

• Chromatin preparation:

  • Crosslinking: Treat S. pombe cells with 1% formaldehyde for 15-20 minutes at room temperature.

  • Cell wall digestion: Use Zymolyase (10 mg/ml) for 30 minutes at 30°C to generate spheroplasts.

  • Chromatin fragmentation: Sonicate to generate 200-500 bp fragments (typically 15-20 cycles of 30 seconds on/30 seconds off).

  • Quality control: Verify fragment size by agarose gel electrophoresis after reversing crosslinks.

• Immunoprecipitation optimization:

  • Pre-clearing: Incubate chromatin with Protein A/G beads for 1 hour to reduce background.

  • Antibody amount: Test multiple antibody quantities (2-10 μg per reaction).

  • Incubation conditions: 4°C overnight with gentle rotation.

  • Washing stringency: Optimize salt concentration in wash buffers (150-500 mM NaCl).

  • Elution method: Direct elution with SDS or on-bead digestion for mass spectrometry.

• Controls for ChIP experiments:

  • Input control: 5-10% of starting chromatin material.

  • Negative control: Non-specific IgG from same species.

  • Positive control: Antibody against histone modifications or known DNA-binding proteins.

  • Technical control: ChIP-qPCR for known target and non-target regions.

• Data analysis considerations:

  • Calculate enrichment relative to input (percent input method).

  • Compare target regions to negative control regions.

  • For ChIP-seq, use peak-calling algorithms with appropriate input normalization.

If SPAC4G9.14 functions as a transcription factor or chromatin-associated protein, validate ChIP results with complementary approaches such as CUT&RUN or ATAC-seq to confirm binding sites.

How can I establish a CRISPR-Cas9 system to study SPAC4G9.14 function in S. pombe?

CRISPR-Cas9 approaches in S. pombe require specific optimizations for efficient genome editing:

• CRISPR-Cas9 system selection:

  • Use SpCas9 (Streptococcus pyogenes Cas9) with optimized codon usage for S. pombe.

  • Consider alternative CRISPR systems (Cas12a/Cpf1) for AT-rich regions.

  • Select expression vectors with appropriate promoters (e.g., nmt1) and selectable markers.

• Guide RNA design:

  • Use S. pombe-specific guide RNA prediction tools that account for PAM availability.

  • Select targets with minimal off-target potential in the S. pombe genome.

  • Target conserved functional domains for knockout studies.

  • For tagging, target sequences near the N- or C-terminus.

  • Design primers for guide validation (e.g., TIDE analysis).

• Donor template design:

  • For knockout: Include selectable marker flanked by 500-1000 bp homology arms.

  • For point mutations: Use single-stranded DNA oligonucleotides (80-120 nt) with the mutation centered.

  • For tagging: Include tag sequence with flexible linker and selection marker.

  • Add silent mutations in the PAM or guide sequence to prevent re-cutting.

• Transformation and screening:

  • Use lithium acetate/PEG method with heat shock for transformation.

  • For transient expression, use a Cas9-2A-sgRNA construct.

  • Include positive selection markers (e.g., G418, hygromycin) for stable integration.

  • Screen transformants by colony PCR, restriction digestion, or sequencing.

  • Validate editing by Western blot using SPAC4G9.14 Antibody.

• Phenotypic characterization:

  • Assess growth rates under standard and stress conditions.

  • Examine cell morphology and division patterns.

  • Analyze protein localization using SPAC4G9.14 Antibody in wild-type versus edited strains.

  • Conduct RNA-seq to identify affected pathways.

Efficiency optimization table: