SCAPER Antibody

What is SCAPER and why is it significant for cell cycle research?

SCAPER is a novel cyclin A-interacting protein that primarily localizes to the endoplasmic reticulum (ER), with a small portion associated with the nucleus. It specifically interacts with cyclin A/Cdk2 in vivo and plays a role in distinguishing S phase from M phase-specific functions of cyclin A/Cdk2 . SCAPER is ubiquitously expressed in human tissues, with highest transcript levels in testis . Its significance lies in its ability to regulate cyclin A localization - ectopic expression of SCAPER sequesters cyclin A from the nucleus and delays cell cycle progression in M phase, while SCAPER ablation decreases the cytoplasmic pool of cyclin A, resulting in delayed progression into S phase from quiescence in response to mitogens .

What is the expression pattern of SCAPER during the cell cycle?

SCAPER is expressed at relatively constant levels throughout the cell cycle, including during quiescence (G0), although it shows slightly elevated levels in early S phase . Semi-quantitative RT-PCR has revealed that SCAPER is expressed at each cell cycle stage, with somewhat higher transcript levels in late G1 and S phase . This expression pattern has been observed at both RNA and protein levels, with the protein detected across various cell lines including immortal lines (T98G, U2OS, HeLa, 293T, Saos2), normal diploid human lines (IMR90, WI38), and mouse myoblast C2C12 cells .

What are the validated applications for SCAPER antibodies?

Currently available SCAPER antibodies have been validated for multiple research applications:

These applications enable researchers to detect, localize, and quantify SCAPER in various experimental contexts, facilitating studies of its biological function and interactions .

What criteria should guide selection of a SCAPER antibody for research?

When selecting a SCAPER antibody, researchers should consider multiple technical aspects:

• Specificity validation: Verify the antibody has been tested against non-target proteins. For example, Novus Biologicals' antibody was validated against a protein array containing the target plus 383 other non-specific proteins .

• Application compatibility: Confirm validation for your intended application(s). Different antibodies are validated for specific techniques - some for multiple applications (Atlas Antibodies ), others for more limited uses (Assay Genie for ELISA and IHC ).

• Immunogen details: Review the immunizing sequence used. For example, Novus Biologicals' antibody was developed against a specific amino acid sequence of SCAPER , while Assay Genie used recombinant human SCAPER protein (amino acids 1109-1400) .

• Host species and antibody type: Consider compatibility with your experimental system. Currently available options include rabbit polyclonal antibodies .

• Recognition of native vs. denatured protein: Ensure the antibody recognizes your protein in its experimental state (folded or denatured).

Given that approximately 50% of commercial antibodies fail to meet basic characterization standards , thorough validation documentation is essential.

How should researchers validate SCAPER antibody specificity?

Comprehensive validation of SCAPER antibody specificity should include:

• Genetic controls: Testing on SCAPER knockdown or knockout samples. As shown in the literature, RNAi-mediated SCAPER ablation has been used to confirm antibody specificity .

• Multi-application testing: Confirming consistent results across different techniques (IHC, ICC-IF, WB) as recommended by antibody characterization experts .

• Peptide competition: Pre-incubating the antibody with immunizing peptide to confirm signal elimination.

• Cross-validation with multiple antibodies: Using antibodies targeting different SCAPER epitopes to confirm consistent results.

• Western blot analysis: Confirming a single band of appropriate molecular weight (~158 kD) .

The NeuroMab approach described in the literature illustrates the rigor required for proper antibody validation, where approximately 1,000 clones are screened using multiple techniques including ELISA, immunohistochemistry, and Western blotting, with emphasis on validating antibodies in their intended applications .

How can SCAPER antibodies be optimized for immunohistochemistry applications?

Optimizing SCAPER antibodies for immunohistochemistry requires systematic parameter adjustment:

• Sample preparation: SCAPER antibodies have been successfully used with paraffin-embedded human tissues, including testis and small intestine .

• Antibody dilution: Begin with the manufacturer's recommended range (e.g., 1:20-1:200 for Assay Genie PACO40506 ), then perform a dilution series to determine optimal signal-to-noise ratio.

• Antigen retrieval: Test multiple methods (heat-induced vs. enzymatic) to maximize epitope accessibility while preserving tissue morphology.

• Detection system: Compare chromogenic (e.g., DAB) and fluorescent detection methods based on your experimental requirements.

• Controls: Include positive controls (tissues with known SCAPER expression, such as testis ) and negative controls (antibody diluent alone, isotype controls).

As emphasized in recent literature, ELISA-based validation alone poorly predicts immunohistochemistry performance, making application-specific optimization critical .

What approaches are effective for studying SCAPER-cyclin A interactions?

Based on published research, several methodological approaches have proven effective for studying SCAPER's interaction with cyclin A/Cdk2:

• Co-immunoprecipitation: Endogenous SCAPER can be immunoprecipitated from cell extracts with anti-cyclin A or anti-Cdk2 antibodies, but not with anti-cyclin E or anti-Cdk1 antibodies . Similarly, immunoprecipitation of Flag-SCAPER co-precipitates cyclin A and Cdk2 .

• Immunofluorescence co-localization: This technique has demonstrated that ectopic SCAPER expression affects endogenous cyclin A localization .

• Cell cycle synchronization studies: Analyzing SCAPER-cyclin A interactions at different cell cycle stages by synchronizing cells through serum deprivation and restimulation .

• Genetic manipulation approaches: Using RNAi to ablate SCAPER expression and examining effects on cyclin A localization and cell cycle progression .

• Subcellular fractionation: Separating cellular compartments to analyze the distribution of SCAPER and cyclin A/Cdk2 complexes, particularly their membrane association .

These approaches can be combined for comprehensive characterization of the functional relationship between SCAPER and cyclin A/Cdk2 complexes.

What buffer conditions preserve SCAPER native structure for immunological detection?

While the search results don't specify optimal buffer conditions for SCAPER antibodies, available product information indicates:

• Storage conditions: SCAPER antibodies are typically stored in PBS, pH 7.2, containing 40% glycerol with 0.02% Sodium Azide or in 50% Glycerol, 0.01M PBS, pH 7.4 with 0.03% Proclin 300 .

• Protein stabilization: The high glycerol content (40-50%) in storage buffers suggests SCAPER antibodies benefit from stabilizing agents to maintain activity.

• Temperature considerations: Short-term storage at 4°C is recommended, with long-term storage at -20°C, avoiding freeze-thaw cycles .

For immunoprecipitation experiments, buffers should be designed to preserve protein-protein interactions while minimizing non-specific binding. When studying SCAPER-cyclin A interactions, researchers should consider that SCAPER is primarily membrane-associated, requiring buffer conditions that maintain membrane protein solubility without disrupting relevant protein complexes .

How can researchers address common technical challenges with SCAPER antibodies?

Researchers working with SCAPER antibodies may encounter several technical challenges that require systematic troubleshooting:

• Weak or absent signal:

  • Verify SCAPER expression in your experimental system (highest in testis, detectable in various cell lines )

  • Optimize antibody concentration based on application (e.g., 1:20-1:200 for IHC )

  • Test different epitope exposure methods (various antigen retrieval techniques)

  • Consider subcellular localization (primarily ER-associated ) when selecting sample preparation methods

• Non-specific background:

  • Increase blocking stringency (longer incubation, different blocking agents)

  • Optimize antibody concentration to minimize non-specific binding

  • Include appropriate controls (isotype control, secondary antibody only)

  • Purify antibodies if necessary (all commercial options are Protein G purified )

• Inconsistent results:

  • Consider batch-to-batch variation, particularly with polyclonal antibodies

  • Standardize protocols across experiments

  • Test multiple antibodies targeting different SCAPER epitopes

• Cross-reactivity concerns:

  • Verify specificity using SCAPER knockdown controls

  • Compare results with antibodies recognizing different SCAPER epitopes

These approaches align with best practices for antibody validation emphasized in recent literature on reproducibility in antibody-based research .

How do researchers reconcile conflicting results between different SCAPER antibodies?

When faced with discordant results from different SCAPER antibodies, researchers should employ a systematic analytical approach:

• Epitope mapping comparison: Different antibodies target distinct regions of SCAPER (e.g., Assay Genie antibody targets amino acids 1109-1400 , while others target different sequences ). Differences in accessibility of these epitopes may explain varying results.

• Validation stringency assessment: Evaluate each antibody's validation profile. More extensively validated antibodies (tested against hundreds of non-target proteins or across multiple applications ) may provide more reliable results.

• Orthogonal method verification: Employ non-antibody-based techniques like mass spectrometry or RNA expression analysis to independently verify protein presence and abundance.

• Genetic controls: Use SCAPER knockdown or knockout models to definitively determine which antibody accurately detects the protein of interest.

• Application-specific optimization: An antibody performing well in one application may fail in another due to differences in how the epitope is presented. As noted in recent literature, "ELISA assays alone may be poor predictors of a reagent useful in other common assays" .

• Post-translational modification sensitivity: Consider whether differences reflect detection of modified forms of SCAPER relevant to its biological function.

Research communities are increasingly emphasizing transparent reporting of both positive and negative outcomes in antibody evaluation to address these challenges .

How can SCAPER antibodies contribute to cell cycle regulation studies?

SCAPER antibodies enable sophisticated investigations into cell cycle regulation through several experimental approaches:

• Temporal regulation analysis: SCAPER antibodies can track protein expression throughout the cell cycle, revealing its relatively constant expression with slight elevation in early S phase .

• Cyclin A/Cdk2 sequestration studies: Immunofluorescence with SCAPER antibodies demonstrates how SCAPER influences cyclin A subcellular localization, potentially sequestering it from nuclear targets .

• Cell cycle transition timing: Using SCAPER antibodies in synchronized cell populations reveals its role in regulating progression from G0 to S phase and through M phase .

• Membrane-cytoplasm-nucleus trafficking: SCAPER antibodies can visualize the protein's distribution between cellular compartments, providing insight into how it regulates cyclin A as a membrane-bound complex .

• Structure-function analysis: By combining SCAPER antibodies with domain-specific mutants, researchers can determine which regions are responsible for cyclin A binding and subcellular localization.

These approaches leverage SCAPER's unique position as a regulator that may distinguish S-phase from M-phase functions of cyclin A/Cdk2 complexes , potentially providing insight into fundamental cell cycle control mechanisms.

What emerging technologies enhance the utility of SCAPER antibodies in research?

Several technological advances can extend the research capabilities of SCAPER antibodies:

• Recombinant antibody production: Moving from traditional polyclonal antibodies to recombinant formats enhances reproducibility and allows for protein engineering. Leading facilities are already converting hybridoma-derived antibodies to recombinant formats with publicly available sequences .

• Super-resolution microscopy: Combining SCAPER antibodies with techniques like STORM, PALM, or SIM could reveal previously undetectable details of its distribution at the ER membrane and its co-localization with cyclin A/Cdk2.

• Proximity labeling approaches: Using SCAPER antibodies in conjunction with BioID or APEX2 systems could identify novel interaction partners beyond cyclin A/Cdk2.

• Single-cell protein analysis: Integrating SCAPER antibodies into mass cytometry (CyTOF) or single-cell Western blotting would enable analysis of protein expression heterogeneity across cell populations.

• Enhanced validation pipelines: Comprehensive approaches similar to NeuroMab's system, where hundreds of clones are screened across multiple applications including ELISA, immunohistochemistry, and Western blotting , could significantly improve SCAPER antibody quality.

These advances address the broader challenge in the antibody field, where approximately 50% of commercial antibodies fail to meet basic characterization standards, resulting in billions of dollars in research waste annually .