SPAPB18E9.05c Antibody

What is the SPAPB18E9.05c protein and why is it studied?

SPAPB18E9.05c is a putative uncharacterized membrane protein found in the fission yeast S. pombe genome. This protein is of interest to researchers studying fundamental cellular processes in this model organism. S. pombe has been extensively utilized in molecular biology research, particularly for understanding cell cycle regulation, DNA damage responses, and basic eukaryotic functions. The SPAPB18E9.05c gene has been cataloged as part of the comprehensive S. pombe genome deletion project, which has classified numerous genes as either essential or viable when deleted. Studying this protein can provide valuable insights into membrane protein function and cellular processes in eukaryotes.

What validated applications exist for the SPAPB18E9.05c antibody?

The SPAPB18E9.05c antibody has been validated for two primary applications:

• Enzyme-Linked Immunosorbent Assay (ELISA): The antibody is effective for ELISA-based detection systems to quantify SPAPB18E9.05c protein in various sample preparations. This allows for sensitive detection and quantification of the target protein in complex mixtures.

• Western Blotting (WB): The antibody enables identification and semi-quantitative analysis of SPAPB18E9.05c protein through Western blot applications. This provides researchers with information about protein expression levels, molecular weight, and potential post-translational modifications.

How is the SPAPB18E9.05c antibody produced?

The SPAPB18E9.05c antibody is produced through a carefully controlled immunization process that follows several key steps:

• Expression and purification of recombinant S. pombe SPAPB18E9.05c protein

• Immunization of rabbits with the purified protein

• Collection of antisera from the immunized animals

• Purification through antigen affinity methods

• Quality control testing to ensure specificity and reactivity

The antigen affinity purification process ensures that only antibodies specific to the SPAPB18E9.05c protein are isolated, which reduces background noise and cross-reactivity in experimental applications. This meticulous production process is essential for creating reliable antibodies for research purposes.

How can epitope mapping be conducted for the SPAPB18E9.05c antibody?

Epitope mapping for SPAPB18E9.05c antibody requires a systematic approach similar to that used for other research antibodies. Based on methodologies described for other antibodies such as those against SARS-CoV-2, researchers can implement the following procedures:

• Peptide Array Analysis: Synthesize overlapping peptides spanning the entire SPAPB18E9.05c protein sequence and test antibody binding to identify specific binding regions.

• Mutational Analysis: Create point mutations in the SPAPB18E9.05c gene and express mutant proteins to determine which amino acid residues are critical for antibody binding, similar to the approach used for identifying critical residues in Spike protein epitopes .

• Competitive Binding Assays: Determine whether other antibodies compete for binding to the same epitope, which would suggest overlapping epitopes as observed in studies with SARS-CoV-2 antibodies .

• Structural Analysis: If resources permit, structural studies such as cryo-electron microscopy could reveal the specific binding interface between the antibody and SPAPB18E9.05c protein, as demonstrated with SARS-CoV-2 antibodies .

Understanding the exact epitope can provide insights into protein function and aid in designing experiments to block or detect specific protein domains.

What are the considerations for using SPAPB18E9.05c antibody in co-immunoprecipitation studies?

When using SPAPB18E9.05c antibody for co-immunoprecipitation (co-IP) studies to identify protein-protein interactions, researchers should consider:

• Antibody Immobilization: Determine the optimal method for immobilizing the antibody to a solid support (e.g., protein A/G beads) while maintaining its binding capacity.

• Membrane Protein Extraction: As SPAPB18E9.05c is described as a membrane protein, special attention must be paid to the lysis conditions. Mild detergents like CHAPS, digitonin, or NP-40 at appropriate concentrations may be necessary to solubilize the protein while preserving protein-protein interactions.

• Control Experiments: Include appropriate negative controls (non-specific IgG) and positive controls (if known interacting partners exist) to validate co-IP results.

• Cross-linking Consideration: For transient or weak interactions, consider using chemical cross-linking agents before cell lysis to stabilize protein complexes.

• Washing Stringency: Optimize washing conditions to remove non-specific binding while retaining specific interactions.

• Elution Methods: Compare different elution methods (low pH, high salt, or competitive elution) to determine which provides the cleanest and most complete recovery of protein complexes.

How can post-translational modifications of SPAPB18E9.05c be studied using the antibody?

Studying post-translational modifications (PTMs) of SPAPB18E9.05c requires specialized approaches:

• Phosphorylation Analysis:

  • Immunoprecipitate SPAPB18E9.05c using the antibody followed by mass spectrometry

  • Use phosphatase treatment on half of the samples to confirm band shifts in Western blots are due to phosphorylation

  • Consider combining with phospho-specific staining methods

• Glycosylation Studies:

  • As a membrane protein, SPAPB18E9.05c may be glycosylated

  • Compare molecular weights before and after treatment with glycosidases

  • Use lectins in combination with the antibody to detect glycosylated forms

• Ubiquitination Detection:

  • Perform immunoprecipitation under denaturing conditions to disrupt protein-protein interactions

  • Probe for ubiquitin in Western blots of immunoprecipitated SPAPB18E9.05c

  • Consider using proteasome inhibitors to enhance detection of ubiquitinated forms

• SUMOylation Analysis:

  • Similar to ubiquitination studies but probing for SUMO proteins

  • Use SUMO-specific proteases to confirm band shifts

Each of these approaches would provide insights into regulatory mechanisms affecting SPAPB18E9.05c function in S. pombe cells.

What is the optimal protocol for using SPAPB18E9.05c antibody in Western blotting?

Based on general antibody practices and the specific information about SPAPB18E9.05c antibody, the following protocol is recommended:

Sample Preparation:

• Harvest S. pombe cells at the appropriate growth phase

• Extract proteins using a suitable lysis buffer containing:

  • 50 mM Tris-HCl, pH 7.5

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

• For membrane proteins like SPAPB18E9.05c, consider adding digitonin (0.5-1%) or CHAPS (0.3-1%)

• Sonicate briefly and centrifuge at 12,000g for 10 minutes at 4°C

• Collect supernatant and quantify protein concentration

Western Blot Procedure:

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

• Transfer to PVDF membrane (0.45 μm pore size recommended for membrane proteins)

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

• Incubate with SPAPB18E9.05c antibody (1:500 to 1:2000 dilution, optimize as needed)

• Wash 3x with TBST

• Incubate with appropriate HRP-conjugated secondary antibody

• Wash 3x with TBST

• Develop using enhanced chemiluminescence (ECL) substrate

• Image using appropriate detection system

Critical Parameters:

• Avoid boiling membrane protein samples; instead, incubate at 37°C for 30 minutes

• Include reducing agent (β-mercaptoethanol) in sample buffer

• Consider gradient gels if the exact molecular weight of the protein is uncertain

How should SPAPB18E9.05c antibody be used in immunofluorescence studies of S. pombe?

Although the available search results don't explicitly mention immunofluorescence as a validated application, researchers interested in this application could adapt the following protocol:

Cell Preparation:

• Grow S. pombe cells to mid-log phase

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

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

• Digest cell walls with Zymolyase-100T (1 mg/ml) in PEM containing 1.2 M sorbitol for 30 minutes at 37°C

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

Immunostaining:

• Block with 5% BSA in PEM for 1 hour

• Incubate with SPAPB18E9.05c antibody (1:100 to 1:200 dilution) overnight at 4°C

• Wash 3x with PEM + 0.1% Tween-20

• Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Wash 3x with PEM + 0.1% Tween-20

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

• Mount and observe under confocal microscope

Controls and Validation:

• Include a secondary-only control

• Consider pre-absorption of the antibody with recombinant SPAPB18E9.05c protein to confirm specificity

• Compare staining pattern with GFP-tagged SPAPB18E9.05c if available

What are the optimal conditions for using SPAPB18E9.05c antibody in ELISA?

For ELISA applications using SPAPB18E9.05c antibody, the following protocol is recommended:

Direct ELISA Protocol:

• Coat wells with purified SPAPB18E9.05c protein or S. pombe cell lysate (10 μg/ml) in carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C

• Wash 3x with PBS-T (PBS with 0.05% Tween-20)

• Block with 3% BSA in PBS-T for 1 hour at room temperature

• Wash 3x with PBS-T

• Add SPAPB18E9.05c antibody serially diluted in PBS-T with 1% BSA

• Incubate for 2 hours at room temperature

• Wash 5x with PBS-T

• Add HRP-conjugated secondary antibody

• Incubate for 1 hour at room temperature

• Wash 5x with PBS-T

• Add TMB substrate and incubate until color develops

• Stop reaction with 2N H₂SO₄

• Read absorbance at 450 nm

Sandwich ELISA (for sample quantification):

• Coat wells with capture antibody (anti-SPAPB18E9.05c or related antibody recognizing a different epitope)

• Block and wash as above

• Add samples and standards

• Add detection antibody (SPAPB18E9.05c antibody, biotinylated if possible)

• Add streptavidin-HRP (if using biotinylated antibody) or appropriate secondary antibody

• Develop and read as above

Optimization Table for ELISA:

How can non-specific binding be reduced when using SPAPB18E9.05c antibody?

Non-specific binding is a common challenge when working with antibodies, especially against less-characterized proteins. The following strategies can help minimize this issue:

• Optimize Blocking Conditions:

  • Test different blocking agents (BSA, milk, casein, commercial blockers)

  • Increase blocking concentration (from 3% to 5%)

  • Extend blocking time (from 1 hour to overnight)

• Antibody Dilution Optimization:

  • Perform titration experiments to find the optimal antibody concentration

  • Consider using higher dilutions (1:2000 to 1:5000) if background is high

• Pre-absorption Treatment:

  • Pre-incubate the antibody with lysates from organisms lacking SPAPB18E9.05c

  • This can remove antibodies that recognize conserved epitopes

• Washing Optimization:

  • Increase washing steps (from 3x to 5x)

  • Extend washing time (from 5 to 10 minutes per wash)

  • Add low concentrations of detergent (0.1-0.5% Triton X-100) to wash buffer

• Additives to Reduce Non-specific Binding:

  • Add 0.1-0.5% Tween-20 to antibody dilution buffer

  • Include 100-200 mM NaCl to reduce ionic interactions

  • Add 0.1% BSA to antibody dilution buffer

• Control Experiments:

  • Include isotype control antibodies

  • Use pre-immune serum as negative control

  • Test the antibody on samples where the target protein is depleted or knocked out

What approach should be taken when contradictory results emerge using SPAPB18E9.05c antibody in different experimental setups?

When faced with contradictory results, a systematic troubleshooting approach is essential:

• Validate Antibody Specificity:

  • Perform Western blot analysis on wild-type vs. SPAPB18E9.05c knockout/knockdown samples

  • Use recombinant SPAPB18E9.05c protein as a positive control

  • Consider peptide competition assays to confirm specificity

• Compare Different Lots and Storage Conditions:

  • Test different antibody lots if available

  • Evaluate proper storage conditions (aliquoting, freeze-thaw cycles)

  • Check for antibody degradation by SDS-PAGE

• Evaluate Experimental Conditions:

  • Create a detailed table comparing all experimental variables between contradictory results

  • Systematically test each variable individually

  • Consider differences in sample preparation, protein extraction methods, or detection systems

• Cross-validate with Alternative Methods:

  • If available, use GFP-tagged SPAPB18E9.05c expression

  • Consider mass spectrometry-based approaches

  • Try RNA-level detection methods (RT-PCR, RNA-seq) to correlate with protein findings

• Biological Variables:

  • Assess cell growth conditions, stress factors, or cell cycle stage

  • Determine if contradictions relate to different cellular compartments

  • Consider post-translational modifications or protein degradation

• Collaborative Verification:

  • Have different researchers or laboratories repeat key experiments

  • Exchange protocols and reagents to identify variables causing discrepancies

How should SPAPB18E9.05c antibody performance be validated for reproducible research?

Ensuring reproducible research with SPAPB18E9.05c antibody requires comprehensive validation steps:

• Initial Characterization:

  • Determine detection limit and dynamic range

  • Assess cross-reactivity with related proteins

  • Confirm epitope specificity through competition assays

• Positive and Negative Controls:

  • Use recombinant SPAPB18E9.05c as positive control

  • Include samples from knockout/knockdown cells as negative controls

  • Test related species to assess cross-species reactivity

• Documentation Requirements:

  • Record complete antibody information:

    • Manufacturer/source

    • Catalog number

    • Lot number

    • Clonality (monoclonal/polyclonal)

    • Host species

    • Immunogen details

  • Document all experimental conditions in detail

• Reproducibility Testing:

  • Perform experiments in multiple biological replicates

  • Test technical reproducibility across different days

  • Have multiple researchers perform key experiments

• Quantitative Validation Metrics:

• Sharing Validation Data:

  • Include validation data in publications supplementary materials

  • Submit antibody validation data to repositories like Antibodypedia

  • Clearly report limitations observed during validation

How can SPAPB18E9.05c antibody be utilized for studying protein localization during cell cycle progression?

S. pombe is a model organism widely used for cell cycle studies. The SPAPB18E9.05c antibody can be applied to investigate protein dynamics throughout the cell cycle:

• Synchronization Experiments:

  • Synchronize S. pombe cultures using methods such as lactose gradient centrifugation, nitrogen starvation, or cdc25-22 temperature-sensitive mutants

  • Collect samples at different cell cycle stages

  • Perform immunofluorescence or cell fractionation followed by Western blotting to track SPAPB18E9.05c localization

• Co-localization Studies:

  • Combine SPAPB18E9.05c antibody with markers for specific cellular compartments:

    • Nucleus (histone proteins)

    • Endoplasmic reticulum (BiP/Kar2)

    • Golgi (Anp1)

    • Plasma membrane (Pma1)

  • Use confocal microscopy to assess co-localization coefficients

• Live Cell Imaging Correlation:

  • Compare fixed-cell antibody staining with live-cell imaging of GFP-tagged SPAPB18E9.05c

  • Create time-lapse series and correlate with cell cycle markers

• Cell Cycle Stage-Specific Extraction:

  • Develop differential extraction protocols to assess protein associations at different cell cycle stages

  • Compare cytoplasmic, membrane, and nuclear fractions

• Data Analysis Approach:

  • Quantify fluorescence intensity changes through the cell cycle

  • Measure co-localization coefficients (Pearson's or Manders')

  • Correlate with cell size or septation index as cell cycle markers

These methods can reveal whether SPAPB18E9.05c shows dynamic localization patterns during cell division, potentially providing insights into its functional role.

What considerations should be made when using SPAPB18E9.05c antibody for chromatin immunoprecipitation (ChIP) experiments?

If there is interest in exploring potential DNA interactions of SPAPB18E9.05c:

• Crosslinking Optimization:

  • Test different formaldehyde concentrations (0.75-1.5%)

  • Evaluate crosslinking times (10-30 minutes)

  • Consider dual crosslinking with disuccinimidyl glutarate (DSG) for protein-protein interactions followed by formaldehyde

• Chromatin Preparation:

  • Optimize sonication conditions for S. pombe cells

  • Aim for DNA fragments between 200-500 bp

  • Verify fragmentation by agarose gel electrophoresis

• Immunoprecipitation Conditions:

  • Test different antibody concentrations

  • Optimize antibody-to-chromatin ratios

  • Compare protein A vs. protein G beads for pulldown

• Controls:

  • Include input DNA control

  • Use IgG from the same species as negative control

  • Consider using GFP-tagged SPAPB18E9.05c with anti-GFP antibody as validation

• Data Analysis:

  • Perform ChIP-qPCR for candidate regions

  • Consider ChIP-seq for genome-wide binding analysis

  • Use appropriate peak-calling algorithms if performing ChIP-seq

• Validation Experiments:

  • Confirm binding sites with EMSAs (Electrophoretic Mobility Shift Assays)

  • Use reporter gene assays to test functional significance of binding

While the search results don't explicitly mention DNA-binding properties for SPAPB18E9.05c, this protocol would be applicable if research indicates potential chromatin association.

How does SPAPB18E9.05c antibody compare with SPAPB18E9.04c antibody in experimental applications?

Based on the search results, both SPAPB18E9.04c and SPAPB18E9.05c antibodies are available as research tools, but direct comparison data is limited. Researchers should consider:

• Sequence Homology Analysis:

  • Examine sequence similarity between SPAPB18E9.04c and SPAPB18E9.05c proteins

  • Higher homology might indicate similar biochemical properties

  • Identify unique domains that might affect antibody performance

• Cross-reactivity Testing:

  • Test each antibody against both recombinant proteins

  • Perform Western blots with both antibodies on the same samples

  • Use peptide competition assays to confirm specificity

• Application Compatibility Comparison:

• Experimental Consistency:

  • Maintain consistent experimental conditions when comparing antibodies

  • Use the same detection systems and sample preparation methods

  • Perform side-by-side experiments to minimize variability

Understanding the similarities and differences between these related antibodies could provide valuable insights into the functions of these neighboring genes in the S. pombe genome.