SPCC1840.07c Antibody

What is SPCC1840.07c and why is it important in research?

SPCC1840.07c is a protein-coding gene in Schizosaccharomyces pombe (fission yeast) that encodes a putative phosphoprotein phosphatase. The gene has an Entrez Gene ID of 2538780 and produces a protein with the accession number NP_588506.1 . Phosphoprotein phosphatases play critical roles in cellular signaling, cell cycle regulation, and stress responses by removing phosphate groups from phosphorylated proteins. Studying SPCC1840.07c is valuable for understanding fundamental eukaryotic cellular processes because S. pombe serves as an excellent model organism with cellular mechanisms conserved across eukaryotes. The phosphatase activity of SPCC1840.07c makes it particularly relevant for research into post-translational modifications and their regulatory roles.

What types of antibodies are available for SPCC1840.07c detection?

Researchers typically have access to several antibody formats for SPCC1840.07c detection:

• Polyclonal antibodies: Generated in host animals (commonly rabbits) immunized with purified recombinant SPCC1840.07c protein or synthetic peptides derived from its sequence. These antibodies recognize multiple epitopes on the target protein, providing strong signal amplification but potentially lower specificity.

• Monoclonal antibodies: Produced from single B-cell clones, offering consistent reproducibility and high specificity for a single epitope, which is advantageous for distinguishing between closely related phosphatases.

• Custom-made antibodies: Available from specialized providers, typically generated against specific peptide sequences within the SPCC1840.07c protein when commercial options are insufficient for particular research applications .

The choice between these antibody types depends on the specific experimental requirements, including detection method sensitivity needs and cross-reactivity concerns with other phosphatases.

How should SPCC1840.07c antibodies be stored and handled to maintain reactivity?

To preserve antibody functionality:

• Store antibodies at -20°C for long-term storage or at 4°C for short-term use (1-2 weeks).

• Avoid repeated freeze-thaw cycles by preparing working aliquots; more than 5 cycles can significantly reduce antibody activity.

• When preparing working dilutions, use sterile buffers containing a carrier protein (0.1-1% BSA) and preservative (0.02% sodium azide).

• Allow antibodies to equilibrate to room temperature before opening the tube to prevent condensation.

• Centrifuge briefly before opening to collect all liquid at the bottom of the vial.

• Document antibody lot numbers, as different lots may show variations in binding characteristics and optimal working concentrations.

Proper storage and handling significantly impact experimental reproducibility and validity of results when working with SPCC1840.07c antibodies.

What are the optimal protocols for Western blot detection of SPCC1840.07c?

For optimal Western blot detection of SPCC1840.07c:

Sample Preparation:

• Lyse S. pombe cells using glass bead disruption in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, and protease/phosphatase inhibitors.

• Clear lysates by centrifugation (14,000 × g, 15 min, 4°C).

• Quantify protein concentration using Bradford or BCA assay.

Electrophoresis and Transfer:

• Load 20-50 μg of total protein per lane on a 10% SDS-PAGE gel.

• Include positive controls from cells overexpressing SPCC1840.07c.

• Transfer to PVDF membrane (0.45 μm) at 100V for 90 minutes in cold transfer buffer.

Detection:

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

• Incubate with primary SPCC1840.07c antibody at 1:1000 dilution overnight at 4°C.

• Wash 3× with TBST, 10 minutes each.

• Incubate with appropriate HRP-conjugated secondary antibody at 1:5000 for 1 hour.

• Wash 3× with TBST, 10 minutes each.

• Develop using ECL substrate and image.

The expected molecular weight of SPCC1840.07c protein is approximately 36.7 kDa. Experimental validation should include both wild-type and knockout/deletion strains to confirm specificity.

How can SPCC1840.07c antibodies be used for immunoprecipitation studies?

For successful immunoprecipitation of SPCC1840.07c:

Protocol:

• Prepare cell lysate in a mild lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 10% glycerol) supplemented with protease and phosphatase inhibitors.

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

• Incubate 1 mg of pre-cleared lysate with 2-5 μg of SPCC1840.07c antibody overnight at 4°C with gentle rotation.

• Add 40 μl of Protein A/G beads and incubate for 3 hours at 4°C.

• Wash beads 4× with lysis buffer.

• Elute proteins by boiling in 2× SDS sample buffer.

• Analyze by Western blot, using a portion of input lysate as control.

Critical considerations:

• Cross-link antibodies to beads with dimethyl pimelimidate (DMP) to prevent antibody co-elution if performing downstream mass spectrometry.

• Include negative controls (non-specific IgG of the same species).

• For phosphatase activity studies, perform immunoprecipitation under native conditions without SDS in buffers.

• When studying SPCC1840.07c interaction partners, consider using formaldehyde cross-linking to capture transient interactions.

This approach allows investigation of protein-protein interactions, post-translational modifications, and enzyme activity of SPCC1840.07c.

How effective are SPCC1840.07c antibodies for immunofluorescence in fission yeast?

Immunofluorescence with SPCC1840.07c antibodies requires specific considerations for yeast cell wall and fixation:

Optimized protocol:

• Grow S. pombe to mid-log phase (OD600 = 0.5-0.8).

• Fix cells with 4% formaldehyde for 30 minutes at room temperature.

• Wash cells in PEM buffer (100 mM PIPES, 1 mM EGTA, 1 mM MgSO4, pH 6.9).

• Digest cell wall with Zymolyase-100T (1 mg/ml) for 30-45 minutes at 37°C.

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

• Block with 5% BSA in PEMBAL buffer for 1 hour.

• Incubate with primary SPCC1840.07c antibody (1:100-1:200) overnight at 4°C.

• Wash 3× with PEMBAL.

• Incubate with fluorophore-conjugated secondary antibody (1:500) for 2 hours.

• Counterstain with DAPI (1 μg/ml) to visualize nuclei.

• Mount and image.

Validation approaches:

• Use SPCC1840.07c deletion strains as negative controls.

• For co-localization studies, include markers for specific subcellular compartments (nucleus, ER, Golgi).

• GFP-tagged SPCC1840.07c strains can serve as positive controls to validate antibody specificity.

The subcellular distribution pattern of SPCC1840.07c can provide insights into its functional roles during different cell cycle stages or under various stress conditions.

How can non-specific binding of SPCC1840.07c antibodies be minimized?

Non-specific binding is a common challenge when working with antibodies against S. pombe proteins. Several strategies can minimize this issue:

For Western blot applications:

• Increase blocking stringency using 5% BSA instead of milk, or add 0.1% Tween-20 to blocking buffer.

• Optimize primary antibody dilution (test range from 1:500 to 1:5000).

• Include competing peptide pre-absorption controls.

• Use PVDF membranes instead of nitrocellulose for potentially better signal-to-noise ratio.

• Perform additional washes with higher salt concentration (up to 500 mM NaCl).

For immunoprecipitation:

• Pre-clear lysates thoroughly with Protein A/G beads.

• Add 0.1-0.5% SDS to wash buffers for more stringent conditions.

• Use monoclonal antibodies when available for higher specificity.

• Consider cross-linking antibodies to beads to prevent heavy chain interference.

For immunofluorescence:

• Extend blocking time to 2 hours.

• Include 0.1% fish skin gelatin in blocking buffer.

• Pre-absorb antibodies with fixed wild-type cells.

• Use confocal microscopy with appropriate filter settings to reduce background.

Performing parallel experiments with SPCC1840.07c knockout strains is the gold standard for distinguishing between specific and non-specific signals.

What strategies help when SPCC1840.07c antibodies show weak or no signal?

When encountering weak or absent signals with SPCC1840.07c antibodies:

For Western blot:

• Increase protein loading (up to 100 μg per lane).

• Extend primary antibody incubation to overnight at 4°C.

• Use enhanced chemiluminescence substrates with higher sensitivity.

• Consider concentrating protein samples using TCA precipitation.

• Test different extraction methods, as SPCC1840.07c may be present in different subcellular fractions.

For immunoprecipitation:

• Scale up starting material (use 2-5 mg of total protein).

• Add protease and phosphatase inhibitors fresh before each experiment.

• Optimize antibody-to-lysate ratio.

• Extend incubation time up to 16 hours.

For immunofluorescence:

• Optimize cell wall digestion time.

• Try different fixation methods (e.g., methanol fixation).

• Use signal amplification systems like tyramide signal amplification.

• Ensure microscope settings are optimized for detection.

If expression levels of endogenous SPCC1840.07c are too low for reliable detection, consider using strains with promoter-enhanced expression or epitope-tagged constructs.

How can specificity of SPCC1840.07c antibodies be verified?

Rigorous validation of antibody specificity is crucial for reliable research findings:

Recommended validation approaches:

• Western blot comparison between wild-type and SPCC1840.07c deletion strains.

• Peptide competition assays where pre-incubation of the antibody with excess immunizing peptide should abolish specific signals.

• Detection of overexpressed SPCC1840.07c protein compared to endogenous levels.

• RNA interference (if applicable) to demonstrate signal reduction upon SPCC1840.07c knockdown.

• Mass spectrometry analysis of immunoprecipitated proteins to confirm target identity.

• Cross-validation using different antibodies targeting distinct epitopes on SPCC1840.07c.

Epitope mapping:
For antibodies generated against the full-length protein, epitope mapping can identify specific recognition sites. This is particularly important when studying protein domains or comparing SPCC1840.07c with related phosphatases.

Complete validation documentation should be maintained for publication purposes and to ensure experimental reproducibility across different antibody lots.

How can SPCC1840.07c antibodies be used to study phosphatase activity and regulation?

SPCC1840.07c antibodies enable several approaches to study phosphatase activity:

Activity assays after immunoprecipitation:

• Immunoprecipitate SPCC1840.07c using optimized protocols.

• Incubate immunoprecipitates with synthetic phosphopeptide substrates.

• Measure released phosphate using malachite green or similar colorimetric assays.

• Include phosphatase inhibitors (okadaic acid, calyculin A) as controls.

Studying regulatory modifications:

• Perform immunoprecipitation under native conditions.

• Analyze immunoprecipitates by western blotting with antibodies against phospho-serine/threonine/tyrosine.

• Use mass spectrometry to identify post-translational modifications on SPCC1840.07c itself.

• Compare phosphatase activity under different cellular conditions (e.g., nutritional stress, cell cycle phases).

Interaction studies:

• Use SPCC1840.07c antibodies for co-immunoprecipitation followed by mass spectrometry.

• Validate interactions with candidate proteins using reverse co-immunoprecipitation.

• Perform proximity ligation assays to visualize protein interactions in situ.

These approaches can reveal how SPCC1840.07c activity is regulated and identify its physiological substrates in fission yeast.

What are the considerations for using SPCC1840.07c antibodies in ChIP applications?

Chromatin immunoprecipitation (ChIP) with SPCC1840.07c antibodies may be valuable if the phosphatase has nuclear functions:

Optimized ChIP protocol for fission yeast:

• Cross-link cells with 1% formaldehyde for 15 minutes.

• Quench with 125 mM glycine.

• Lyse cells using glass beads.

• Sonicate chromatin to 200-500 bp fragments.

• Pre-clear chromatin with Protein A/G beads.

• Immunoprecipitate with SPCC1840.07c antibody overnight.

• Wash extensively with increasingly stringent buffers.

• Reverse cross-links and purify DNA.

• Analyze by qPCR or sequencing.

Critical considerations:

• Validate antibody specificity in ChIP conditions using deletion strains.

• Include appropriate controls (input, IgG, positive control antibody).

• Optimize sonication conditions for consistent fragmentation.

• Consider dual cross-linking with ethylene glycol bis(succinimidyl succinate) for improved protein-protein cross-linking.

While phosphatases are not typically DNA-binding proteins, SPCC1840.07c may associate with chromatin through interactions with transcription factors or chromatin modifiers. ChIP experiments could reveal roles in transcriptional regulation or DNA damage response.

How can SPCC1840.07c antibodies be integrated into systems biology approaches?

SPCC1840.07c antibodies can support systems-level investigations:

Multi-omics integration strategies:

• Combine immunoprecipitation with mass spectrometry to define the SPCC1840.07c interactome under various conditions.

• Correlate changes in the phosphoproteome (using phospho-specific antibodies or phosphoproteomics) with SPCC1840.07c activity.

• Integrate with transcriptomic data to identify genes affected by SPCC1840.07c function.

• Develop computational models of phosphorylation/dephosphorylation networks incorporating SPCC1840.07c.

Comparative studies across species:
The following table shows homologs of SPCC1840.07c in other model organisms for comparative studies:

Using antibodies against these homologs in parallel experiments can reveal evolutionarily conserved functions and regulatory mechanisms of this phosphatase family.

High-throughput screening applications:

• Develop immunofluorescence-based screens in S. pombe to identify compounds affecting SPCC1840.07c localization or expression.

• Use antibody-based protein arrays to study phosphatase-substrate relationships systematically.

• Implement automated image analysis pipelines for quantifying SPCC1840.07c dynamics in response to perturbations.

These systems approaches can position SPCC1840.07c within broader signaling networks and reveal its contributions to cellular homeostasis.

How can SPCC1840.07c antibodies be used with super-resolution microscopy?

Super-resolution microscopy offers unprecedented insights into protein localization:

Optimized approaches:

• For STORM/PALM: Use primary SPCC1840.07c antibodies with photoswitchable fluorophore-conjugated secondary antibodies.

• For SIM: Standard immunofluorescence protocols can be applied, with attention to increased signal requirements.

• For STED: Use secondary antibodies conjugated with STED-compatible dyes (e.g., STAR635P, ATTO647N).

Technical considerations:

• Cell wall digestion must be carefully optimized to maintain structural integrity while allowing antibody access.

• Sample mounting media should match the refractive index requirements of the microscopy technique.

• Multi-color imaging requires careful selection of fluorophores with minimal spectral overlap.

• Quantitative analysis should include proper controls for background subtraction and drift correction.

Super-resolution imaging can reveal precise subcellular localization patterns of SPCC1840.07c that might be missed by conventional microscopy, potentially identifying specific foci or exclusion from certain cellular compartments.

What are the considerations for developing proximity-dependent labeling using SPCC1840.07c antibodies?

Proximity-dependent labeling can map the protein neighborhood of SPCC1840.07c:

BioID or APEX2-based approaches:

• Generate fusion constructs of SPCC1840.07c with BioID2 or APEX2.

• Express these constructs in S. pombe.

• Activate labeling with biotin or biotin-phenol/H₂O₂.

• Purify biotinylated proteins using streptavidin beads.

• Identify interacting proteins by mass spectrometry.

Antibody-based proximity labeling:

• Conjugate SPCC1840.07c antibodies with HRP or APEX2.

• Apply to fixed cells or cell lysates.

• Activate labeling with biotin-tyramide/H₂O₂.

• Analyze biotinylated proteins by streptavidin pulldown followed by Western blot or mass spectrometry.

These techniques can identify weak or transient interactions missed by conventional co-immunoprecipitation and provide spatial context for SPCC1840.07c function within the cell.

How can multiplexed detection methods be applied with SPCC1840.07c antibodies?

Modern multiplexed detection enables simultaneous analysis of multiple proteins:

Mass cytometry (CyTOF) approaches:

• Label SPCC1840.07c antibodies with rare earth metals.

• Combine with antibodies against other proteins of interest, each labeled with different metals.

• Analyze by mass cytometry for single-cell quantification.

Sequential immunofluorescence:

• Perform immunofluorescence with SPCC1840.07c antibodies.

• Image the sample.

• Strip antibodies using glycine-HCl buffer (pH 2.5).

• Reprobe with antibodies against different targets.

• Repeat imaging and computational alignment.

Multiplex Western blotting:

• Use fluorescently labeled secondary antibodies with distinct spectra.

• Image on multi-channel fluorescence scanners.

• Alternatively, use sequential reprobing with antibody stripping between rounds.

These multiplexed approaches allow researchers to study SPCC1840.07c in the context of complex signaling networks and cellular processes, providing a more comprehensive understanding of its functions and regulations.