SPAC19B12.07c Antibody

What is SPAC19B12.07c and why is it studied in Schizosaccharomyces pombe?

SPAC19B12.07c is a zinc finger protein found in Schizosaccharomyces pombe (fission yeast) . Zinc finger proteins play critical roles in transcription regulation, DNA recognition, RNA packaging, and protein-protein interactions. Studying this protein in S. pombe is valuable because this organism serves as an excellent model system with cellular processes similar to those in higher eukaryotes, including humans, while maintaining a relatively simple genome structure .

What types of SPAC19B12.07c antibodies are available for research?

The primary antibody available for SPAC19B12.07c research is a rabbit polyclonal antibody generated against recombinant Schizosaccharomyces pombe (strain 972/ATCC 24843) SPAC19B12.07c protein . This antibody is:

How should SPAC19B12.07c antibody be stored for optimal performance?

For short-term storage, keep the antibody at 4°C. For long-term storage, store at -20°C to -80°C . Aliquot the antibody into smaller volumes to avoid repeated freeze-thaw cycles, which can degrade the antibody and reduce its effectiveness. Each freeze-thaw cycle can decrease antibody activity by 10-15%, significantly impacting experimental results over time.

What is the recommended protocol for Western blotting with SPAC19B12.07c antibody?

For optimal Western blotting results with SPAC19B12.07c antibody:

• Sample preparation: Lyse S. pombe cells using glass bead disruption in appropriate buffer (e.g., 50mM Tris-HCl pH 7.5, 150mM NaCl, 0.5% NP-40, protease inhibitors)

• Protein separation: Run 20-50μg total protein per lane on 10-12% SDS-PAGE

• Transfer: Transfer proteins to PVDF or nitrocellulose membrane using standard methods

• Blocking: Block with 5% non-fat milk in TBST for 1 hour at room temperature

• Primary antibody: Dilute SPAC19B12.07c antibody 1:5000 in blocking solution; incubate overnight at 4°C

• Washing: Wash 3× with TBST, 5 minutes each

• Secondary antibody: Anti-rabbit HRP-conjugated secondary antibody (1:5000-1:10000); incubate 1 hour at room temperature

• Detection: Use ECL substrate and appropriate imaging system

Note that optimization may be required for your specific experimental conditions and cell preparation method.

How can I validate the specificity of SPAC19B12.07c antibody in my experiments?

Validating antibody specificity is critical for reliable results. Consider these approaches:

• Positive control: Use purified recombinant SPAC19B12.07c protein or lysates from wild-type S. pombe

• Negative control: Include lysates from SPAC19B12.07c deletion mutants if available

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide before application

• Immunoprecipitation followed by mass spectrometry: Confirm the identity of the precipitated protein

• Cross-reactivity testing: Test reactivity with closely related proteins to determine specificity

Using multiple validation methods provides stronger evidence for antibody specificity than relying on a single approach.

What factors influence the sensitivity of ELISA assays using SPAC19B12.07c antibody?

Several factors can affect ELISA sensitivity when using SPAC19B12.07c antibody:

Following standardized ELISA protocols, such as those outlined by WHO reference laboratories , and optimizing each step for your specific experimental setup will help maximize sensitivity and reproducibility.

How can I troubleshoot weak or absent signal when using SPAC19B12.07c antibody?

When encountering weak or absent signals with SPAC19B12.07c antibody:

• Antibody activity: Check storage conditions; consider obtaining a new lot if the antibody has undergone multiple freeze-thaw cycles

• Protein expression level: SPAC19B12.07c may be expressed at low levels under standard conditions; consider enrichment techniques

• Epitope accessibility: The protein conformation may obscure the epitope; try different lysis and denaturation conditions

• Protein transfer efficiency: Ensure complete transfer to the membrane; use staining to verify transfer

• Detection sensitivity: Use more sensitive detection reagents (enhanced chemiluminescence substrates)

• Exposure time: Increase camera exposure time or film exposure for weaker signals

• Protein degradation: Include additional protease inhibitors in your lysis buffer

• Antibody concentration: Decrease the antibody dilution (use more concentrated antibody solution)

Systematic troubleshooting by changing one variable at a time will help identify the source of the problem.

How can SPAC19B12.07c antibody be used to study protein-protein interactions?

To investigate protein-protein interactions involving SPAC19B12.07c:

• Co-immunoprecipitation (Co-IP):

  • Lyse cells in non-denaturing buffer to preserve protein complexes

  • Immunoprecipitate with SPAC19B12.07c antibody

  • Analyze co-precipitated proteins by mass spectrometry or Western blot

• Proximity ligation assay (PLA):

  • Use SPAC19B12.07c antibody in combination with antibodies against suspected interaction partners

  • Apply oligonucleotide-linked secondary antibodies

  • Detect protein proximity (≤40nm) through rolling circle amplification

• Chromatin immunoprecipitation (ChIP):

  • If SPAC19B12.07c functions in DNA binding, use the antibody to identify genomic binding sites

  • Follow standard ChIP protocols with appropriate modifications for S. pombe

• Bimolecular fluorescence complementation (BiFC):

  • Generate fusion proteins with split fluorescent protein fragments

  • Use the antibody to confirm expression of the fusion proteins

Each method provides different insights into protein interactions and should be selected based on your specific research questions.

How does SPAC19B12.07c antibody compare to other S. pombe protein antibodies in terms of detection sensitivity?

Comparative studies of antibody performance in S. pombe show variability in detection sensitivity:

• Anti-Mrc1 antibodies typically show high specificity with detection of phosphorylated forms in Western blots at dilutions of 1:5,000, allowing visualization of mobility shifts caused by phosphorylation events .

• SAPK/JNK antibodies demonstrate cross-reactivity with S. cerevisiae proteins at 46 and 54 kDa molecular weights, with high sensitivity for endogenous protein levels .

• SPAC19B12.07c antibody performance depends on expression levels, which may vary under different experimental conditions.

For optimal comparative studies, standardize protein extraction methods, loading amounts, and detection systems across different antibodies. Consider using dual-color Western blot systems to directly compare antibody performance on the same membrane when possible.

How should I normalize SPAC19B12.07c protein expression data in comparative studies?

For reliable quantification of SPAC19B12.07c expression:

• Internal loading controls:

  • Use antibodies against stable housekeeping proteins (e.g., actin, GAPDH, tubulin)

  • For S. pombe specifically, Cdc2 or α-tubulin are often used as reference proteins

• Total protein normalization:

  • Stain membranes with total protein stains (Ponceau S, SYPRO Ruby)

  • Calculate the ratio of target band intensity to total protein signal

• Multiple reference genes approach:

  • Use at least two different reference proteins

  • Calculate geometric means of reference signals for more robust normalization

• Statistical analysis:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests (e.g., t-test, ANOVA) depending on your experimental design

  • Report both raw and normalized data with appropriate measures of variation

The choice of normalization method should be based on your experimental conditions, as reference protein expression may also change under certain treatments.

What are the challenges in interpreting contradictory results from different SPAC19B12.07c antibody detection methods?

When facing contradictory results between different detection methods:

• Different epitope recognition:

  • Western blotting detects denatured epitopes

  • ELISA may detect native or denatured epitopes depending on the assay design

  • Immunoprecipitation recognizes native conformations

• Post-translational modifications (PTMs):

  • Some antibodies may be sensitive to PTMs that mask or expose epitopes

  • Different methods may preserve or disrupt these modifications

• Cross-reactivity profiles:

  • Each antibody preparation may have unique cross-reactivity patterns

  • Validation in knockout strains is critical for confirming specificity

• Resolution approach:

  • Use multiple antibodies targeting different epitopes of SPAC19B12.07c

  • Employ orthogonal detection methods (e.g., mass spectrometry)

  • Consider genetic approaches (epitope tagging) for validation

When publishing contradictory results, clearly report all experimental conditions, antibody information (including lot number), and validation steps performed.

How can SPAC19B12.07c antibody contribute to understanding sarbecovirus neutralizing antibody development?

Recent research on sarbecovirus neutralizing antibodies provides insights into how SPAC19B12.07c studies might contribute to broader immunological understanding:

• Structural similarity analyses: Studies of non-immunodominant epitopes in SARS-CoV-2 revealed that antibodies targeting conserved sites associated with critical viral functions are less likely to be escaped by mutations . Similar approaches could be applied to analyze SPAC19B12.07c epitope conservation across related proteins.

• Longevity of antibody response: Research on SARS-CoV-2 showed that neutralizing antibodies reached a plateau after 2 weeks but then declined in most patients . This temporal pattern analysis methodology could be applied to study the dynamics of antibodies produced against SPAC19B12.07c in various experimental contexts.

• Cross-reactivity assessment: Methods used to determine specificity of anti-SARS-CoV-2 antibodies, which showed minimal cross-reactivity with other coronaviruses , could be adapted to evaluate the specificity of SPAC19B12.07c antibodies against related zinc finger proteins.

• Validation through multiple detection methods: The combinatorial approach of using both binding assays (ELISA) and functional assays (neutralization) to validate SARS-CoV-2 antibodies demonstrates the importance of using multiple methods to comprehensively characterize SPAC19B12.07c antibodies.

What genomic screening approaches can be enhanced using SPAC19B12.07c antibody?

Genomic screening methodologies using S. pombe can be enhanced with SPAC19B12.07c antibody:

• Fitness profiling in nutrient-altered environments: Building on work that identified genes regulating fitness in response to nutrient changes , SPAC19B12.07c antibody could help track protein expression changes in different nutritional conditions.

• Antifungal drug sensitivity screens: Following the methodology of genomewide screens for genes affecting sensitivity to antifungal drugs , SPAC19B12.07c antibody could be used to determine if protein levels correlate with drug sensitivity phenotypes.

• Chromatin organization studies: Given that zinc finger proteins often function in chromatin regulation, SPAC19B12.07c antibody could be employed in ChIP-seq experiments to map genomic binding sites and identify target genes.

• Proteome-wide interaction mapping: Using approaches similar to those that identified broad sarbecovirus-neutralizing antibody interactions , immunoprecipitation with SPAC19B12.07c antibody followed by mass spectrometry could reveal novel protein interaction networks.

• CRISPR-based functional genomics: The antibody could validate protein depletion in CRISPR screens designed to identify genetic interactions with SPAC19B12.07c.

By integrating these approaches, researchers can develop a more comprehensive understanding of SPAC19B12.07c function in cellular processes.