CYCH1-1 Antibody

What is the CYCH1-1 antibody and what does it target?

The CYCH1-1 antibody is a specific antibody designed to recognize and bind to cyclin H;1 (CycH;1), which is a regulatory protein involved in cell cycle progression. This antibody has been particularly useful in plant research, where it can detect multiple forms of CycH;1 protein ranging from 37 to 40 kDa. The antibody binds to epitopes on the CycH;1 protein, enabling researchers to study its expression, localization, and interactions with other proteins such as cyclin-dependent kinases (CDKs). In Arabidopsis research, the antibody has been crucial for investigating phosphoregulatory mechanisms controlling cyclin-dependent protein kinases .

What are the primary applications of CYCH1-1 antibody in research?

The CYCH1-1 antibody has several important applications in research:

• Immunoprecipitation: Used to isolate and concentrate CycH;1 and its associated proteins from cell extracts

• Western blotting: Detection of CycH;1 protein in cell or tissue lysates

• Immunohistochemistry: Localization of CycH;1 in tissue sections

• Protein complex analysis: Investigation of CycH;1 interactions with other proteins, particularly cyclin-dependent kinases

• Cellular fractionation studies: Analysis of CycH;1 distribution in subcellular compartments

These applications have been documented in studies examining cell cycle regulation in plant systems, where the antibody can detect three distinct bands of CycH;1 at 37, 39, and 40 kDa when used for immunoblotting of Arabidopsis extracts .

How should CYCH1-1 antibody be validated before use in experiments?

Proper validation is critical for ensuring reliable research outcomes when using CYCH1-1 antibody:

• Specificity testing: Confirm that the antibody specifically recognizes CycH;1 through depletion experiments. For example, researchers have verified specificity by depleting the antibody from antiserum using Ni-NTA agarose carrying His-CycH;1, which resulted in the disappearance of detected bands on immunoblots .

• Positive and negative controls: Include appropriate positive controls (samples known to express CycH;1) and negative controls (samples without CycH;1 expression) in experiments.

• Cross-reactivity assessment: Test against similar proteins to ensure specificity, particularly other cyclin family members.

• Application-specific validation: Validate the antibody separately for each application (Western blot, immunoprecipitation, etc.) as performance can vary between applications .

• Knockout/knockdown verification: When possible, test against samples where CycH;1 has been deleted or reduced to confirm specificity .

These validation steps align with recommended practices for antibody characterization described in scientific literature, which note that approximately 50% of commercial antibodies fail to meet basic standards for characterization .

What are the optimal conditions for using CYCH1-1 antibody in immunoprecipitation experiments?

For optimal immunoprecipitation experiments with CYCH1-1 antibody:

• Sample preparation: Use fresh protein extracts (200-400 μg) from your biological material. For plant tissues like Arabidopsis, specialized extraction buffers containing protease inhibitors are recommended.

• Antibody concentration: Based on published protocols, use approximately 2-5 μg of CYCH1-1 antibody per 200 μg of protein extract.

• Incubation conditions: Incubate the antibody with the protein extract for 2-4 hours at 4°C with gentle rotation.

• Capture method: Use protein A/G beads or similar matrices to capture the antibody-protein complexes.

• Washing stringency: Multiple washes with decreasing salt concentrations help reduce non-specific binding while preserving specific interactions.

Research has shown that this approach can successfully recover all three CycH;1 protein forms (37, 39, and 40 kDa) from Arabidopsis extracts, allowing for downstream analysis of associated proteins such as CDKD;2 and CDKD;3 .

How should researchers optimize Western blotting protocols for CYCH1-1 antibody?

Optimized Western blotting with CYCH1-1 antibody requires:

• Sample preparation:

  • Use 15-20 μg of total protein for plant samples

  • Include denaturing agents and reducing conditions in sample buffer

  • Fresh samples yield better results than stored samples

• Gel parameters:

  • 10-12% SDS-PAGE gels offer optimal resolution for CycH;1 proteins (37-40 kDa range)

  • Consider gradient gels (8-16%) when analyzing complexes

• Transfer conditions:

  • Semi-dry transfer: 15V for 30 minutes

  • Wet transfer: 100V for 1 hour or 30V overnight at 4°C

  • PVDF membranes typically work better than nitrocellulose for CycH;1 detection

• Blocking and antibody dilutions:

  • 5% non-fat dry milk or 3% BSA in TBST for blocking (1 hour at room temperature)

  • Primary antibody dilution: 1:1000 to 1:2000 (incubate overnight at 4°C)

  • Secondary antibody dilution: 1:5000 to 1:10000 (incubate 1 hour at room temperature)

• Detection system:

  • Enhanced chemiluminescence (ECL) provides sufficient sensitivity

  • Exposure times typically range from 30 seconds to 5 minutes

These recommendations are based on published protocols that have successfully detected CycH;1 proteins in Arabidopsis extracts .

What controls should be included when using CYCH1-1 antibody in research?

Comprehensive controls are essential for reliable results:

• Antibody specificity controls:

  • Antibody depletion using recombinant target protein (e.g., His-CycH;1 bound to Ni-NTA agarose)

  • Pre-immune serum control (when available)

  • Isotype-matched control antibody

• Sample-related controls:

  • Positive control: Sample known to express CycH;1 (e.g., Arabidopsis root tissue)

  • Negative control: Sample with minimal/no CycH;1 expression

  • Knockout/knockdown samples (when available)

• Procedural controls:

  • Loading control (e.g., GAPDH, actin, tubulin) for Western blots

  • Input sample aliquot for immunoprecipitation experiments

  • Secondary antibody-only control

• Validation across applications:

  • Cross-validation using different detection methods

  • Comparing results across different antibody clones/sources when possible

Research has shown that CycH;1 expression varies between plant tissues, with higher expression in roots compared to shoots, making tissue-specific controls important .

How can CYCH1-1 antibody be used to study protein complexes and interactions?

Advanced protein complex analysis with CYCH1-1 antibody can be conducted through:

• Sequential immunoprecipitation:

  • First immunoprecipitate with CYCH1-1 antibody

  • Elute and perform second immunoprecipitation with antibodies against suspected interaction partners

  • This approach has successfully identified CycH;1-CDKD complexes in Arabidopsis

• Size exclusion chromatography combined with immunoblotting:

  • Fractionate protein extracts using columns like Sephacryl S300

  • Analyze fractions by immunoblotting with CYCH1-1 antibody

  • Research has shown CycH;1 protein distribution across fractions from 50-250 kDa, overlapping with CDKD;2 and CDKD;3 distribution

• In vitro kinase assays of immunoprecipitated complexes:

  • Immunoprecipitate CycH;1 complexes

  • Test kinase activity using appropriate substrates (e.g., GST-CDK2 or GST-CTD)

  • This approach has confirmed the functionality of precipitated complexes

• Co-localization studies:

  • Combine CYCH1-1 antibody with antibodies against potential interaction partners

  • Use fluorescently-labeled secondary antibodies for detection

  • Analyze co-localization using confocal microscopy

These approaches have revealed that different forms of CycH;1 (37 kDa vs. 39 kDa) may preferentially interact with different CDKD proteins, suggesting functional specialization .

What are the considerations for using CYCH1-1 antibody in immunohistochemistry and immunofluorescence?

For optimal immunohistochemistry and immunofluorescence with CYCH1-1 antibody:

• Fixation methods:

  • Paraformaldehyde (4%) is generally effective for plant tissues

  • Fixation time should be optimized (typically 20-30 minutes)

  • Overfixation can mask epitopes and reduce antibody binding

• Antigen retrieval:

  • Heat-induced epitope retrieval (citrate buffer, pH 6.0)

  • Enzymatic retrieval methods may be necessary for some tissues

  • Test different retrieval methods to determine optimal conditions

• Blocking and permeabilization:

  • Block with 5% normal serum from the species of secondary antibody

  • Include 0.1-0.3% Triton X-100 for cell permeabilization

  • Longer blocking times (2+ hours) may reduce background

• Antibody dilutions and incubation:

  • Primary antibody: 1:50 to 1:200 dilution

  • Incubate overnight at 4°C for best results

  • Secondary antibody: 1:200 to 1:500 dilution

• Detection systems:

  • For immunofluorescence: fluorophore-conjugated secondary antibodies

  • For immunohistochemistry: HRP or AP-based detection systems

Researchers have successfully used GFP-tagged CycH;1 for localization studies in tobacco BY2 cells, providing a complementary approach to antibody-based detection .

How can differences in CycH;1 protein forms (37, 39, and 40 kDa) be characterized using the CYCH1-1 antibody?

Comprehensive characterization of different CycH;1 forms requires:

• High-resolution gel electrophoresis:

  • Use long separation gels (15-20 cm) with 10-12% acrylamide

  • Consider 2D gel electrophoresis to separate based on both molecular weight and isoelectric point

  • Longer running times at lower voltage improve separation

• Phosphorylation-specific analysis:

  • Treat samples with phosphatases before immunoblotting

  • Use Phos-tag™ acrylamide gels for enhanced separation of phosphorylated forms

  • Apply phospho-specific stains in parallel with immunoblotting

• Mass spectrometry of immunoprecipitated proteins:

  • Immunoprecipitate with CYCH1-1 antibody

  • Excise specific bands for mass spectrometry analysis

  • Identify post-translational modifications and sequence variations

• Differential extraction methods:

  • Compare protein profiles from different subcellular fractions

  • Use varying salt concentrations to extract differentially associated proteins

  • Research has shown the 37 kDa CycH;1 form may associate with both CDKD;2 and CDKD;3, while the 39 kDa form preferentially associates with CDKD;2

• Functional analysis of separated complexes:

  • Use size exclusion chromatography to separate complexes

  • Test kinase activity of different fractions against substrates like GST-CTD

  • Correlate activity with presence of specific CycH;1 forms

Research suggests the different forms may represent post-translationally modified variants with distinct binding properties and functional roles in cyclin-dependent kinase complexes .

What are common issues when using CYCH1-1 antibody and how can they be resolved?

Research has shown that the CYCH1-1 antibody may recognize different forms of CycH;1 with varying affinity, which can affect experimental outcomes .

How should researchers quantify and report CYCH1-1 antibody-based results?

For reliable quantification and reporting:

• Western blot quantification:

  • Use digital image analysis software (ImageJ, ImageLab, etc.)

  • Include a standard curve of recombinant protein when possible

  • Always normalize to appropriate loading controls

  • Report relative rather than absolute values

• Statistical considerations:

  • Perform at least three biological replicates

  • Apply appropriate statistical tests (t-test, ANOVA)

  • Include error bars representing standard deviation or standard error

  • Report exact p-values rather than thresholds

• Control inclusion and reporting:

  • Always show representative blots including molecular weight markers

  • Include images of appropriate negative and positive controls

  • Show full blots in supplementary materials

  • Disclose any image adjustments applied (contrast, brightness)

• Antibody documentation:

  • Report antibody source, catalog number, and lot number

  • Describe validation methods used

  • Detail all experimental conditions (dilutions, incubation times)

  • Follow antibody reporting guidelines as recommended by scientific societies

These practices align with recommendations for improving reproducibility in antibody-based research, addressing issues that have led to estimated financial losses of $0.4-1.8 billion per year due to poorly characterized antibodies .

How can researchers assess batch-to-batch variation in CYCH1-1 antibody preparations?

To manage antibody batch variation:

• Standardized testing protocol:

  • Test each new batch against a standard positive control sample

  • Run side-by-side comparison with previous batch

  • Document detection sensitivity, specificity, and background

• Quantitative assessment:

  • Measure signal-to-noise ratio across batches

  • Determine minimal detection threshold for each batch

  • Calculate coefficient of variation between batches

• Performance metrics to evaluate:

  • Epitope recognition pattern (single vs. multiple bands)

  • Dilution curve comparison

  • Background levels in negative controls

  • Immunoprecipitation efficiency

• Record-keeping system:

  • Maintain detailed inventory with batch numbers

  • Document performance characteristics of each batch

  • Link experimental results to specific antibody batches

• Alternative approaches:

  • Consider recombinant antibody technology for improved consistency

  • Sequence antibody variable regions when possible

  • Develop internal reference standards

Initiatives like NeuroMab have developed approaches to convert monoclonal antibodies into recombinant forms and make their sequences publicly available, which could serve as a model for improving consistency in CYCH1-1 antibody research .

How can CYCH1-1 antibody be used in combination with other techniques for comprehensive protein characterization?

Integrative approaches using CYCH1-1 antibody can provide deeper insights:

• Combinatorial immunoprecipitation with mass spectrometry:

  • Use CYCH1-1 antibody for initial protein complex isolation

  • Analyze precipitated proteins by LC-MS/MS

  • Apply label-free quantification or SILAC for comparative studies

  • This approach can identify novel interaction partners beyond known CDKDs

• ChIP-seq with CYCH1-1 antibody:

  • Apply chromatin immunoprecipitation to identify genomic binding sites

  • Sequence precipitated DNA fragments

  • Map potential regulatory roles of CycH;1 in transcription

• Proximity labeling approaches:

  • Combine with BioID or APEX2 proximity labeling

  • Identify proteins in close proximity to CycH;1

  • Compare interactome across different cellular conditions

• Live-cell imaging with complementary fluorescent protein approaches:

  • Use antibody validation in fixed cells alongside GFP-tagged CycH;1 in live cells

  • Correlate antibody staining patterns with live-cell dynamics

  • Studies have successfully used CycH;1-GFP for localization in tobacco BY2 cells

• Single-cell analysis:

  • Apply CYCH1-1 antibody in single-cell Western blotting

  • Use for CyTOF mass cytometry to analyze expression in heterogeneous populations

  • Combine with single-cell transcriptomics for multi-omics integration

These integrated approaches align with recent trends in antibody-based research that emphasize multi-method validation and characterization .

What are the considerations for developing improved versions of CYCH1-1 antibody?

For antibody improvement strategies:

• Epitope mapping and optimization:

  • Identify the specific epitope(s) recognized by current CYCH1-1 antibody

  • Design new immunogens targeting highly specific regions

  • Consider conserved vs. variable regions depending on research needs

• Conversion to recombinant format:

  • Sequence variable regions of existing antibody

  • Express as recombinant antibody fragments (scFv, Fab)

  • Introduce affinity-enhancing mutations if needed

  • This approach has been successfully used by initiatives like NeuroMab

• Format diversification:

  • Develop directly labeled primary antibodies

  • Create bispecific antibodies for co-detection of interacting partners

  • Generate nanobody alternatives for improved tissue penetration

• Validation strategy design:

  • Implement multi-assay validation pipeline

  • Test against knockout/knockdown controls

  • Evaluate performance in all intended applications

  • Follow guidelines similar to those used by the Protein Capture Reagents Program

• Data sharing and standardization:

  • Document complete validation data

  • Share antibody sequences when possible

  • Deposit standardized protocols in repositories

These approaches reflect best practices from antibody development initiatives that emphasize rigorous characterization and validation .

What emerging technologies might complement or replace CYCH1-1 antibody-based detection methods?

Emerging technologies with potential impact include:

• Aptamer-based detection systems:

  • DNA/RNA aptamers selected against CycH;1

  • Potentially higher specificity and reproducibility

  • Compatible with live-cell applications

• CRISPR-based tagging:

  • Endogenous tagging of CycH;1 with epitope tags or fluorescent proteins

  • Eliminates antibody specificity concerns

  • Enables live-cell tracking of native protein

• Mass spectrometry-based targeted proteomics:

  • Develop SRM/MRM assays for CycH;1 quantification

  • Absolute quantification without antibodies

  • Higher specificity for distinguishing protein forms

• Nanobody and single-domain antibody alternatives:

  • Smaller size enables better tissue penetration

  • Potentially higher stability and specificity

  • Easier recombinant production

• Microfluidic and single-molecule detection platforms:

  • Higher sensitivity detection systems

  • Reduced sample requirements

  • Potential for multiplexed analysis

These technologies align with trends toward more reproducible, quantitative methods in protein research, addressing some limitations of traditional antibodies highlighted in recent reviews .

How should researchers approach contradictory or unexpected results when using CYCH1-1 antibody?

When facing unexpected results:

• Systematic validation approach:

  • Verify antibody specificity using depletion controls

  • Test multiple antibody dilutions and incubation conditions

  • Compare results across different biological replicates

  • Apply orthogonal detection methods when possible

• Common sources of contradictory results:

  • Post-translational modifications affecting epitope recognition

  • Protein complex formation masking antibody binding sites

  • Tissue-specific expression of protein isoforms

  • Technical variations in sample preparation

• Documentation and reporting strategy:

  • Record all experimental conditions in detail

  • Document unexpected findings thoroughly

  • Compare with published literature on CycH;1

  • Consider publishing negative or contradictory results

• Collaborative verification:

  • Share samples and protocols with collaborating laboratories

  • Test the same samples with alternative antibodies

  • Consider round-robin testing approaches

• Technical considerations:

  • Examine the effect of different lysis buffers on protein extraction

  • Test native versus denaturing conditions

  • Evaluate the impact of sample storage conditions

Research has shown that CycH;1 exists in multiple forms with potentially different binding partners and functions, which may explain apparently contradictory results in different experimental settings .

What are the key considerations for researchers transitioning from model systems to clinical samples when using CYCH1-1 antibody?

For translational research applications:

• Species cross-reactivity assessment:

  • Validate antibody recognition of human CycH;1 homologs

  • Compare epitope conservation across species

  • Test on matched samples from different species

• Clinical sample preparation optimization:

  • Develop modified protocols for FFPE or frozen clinical samples

  • Optimize antigen retrieval methods for tissue sections

  • Establish standardized processing for blood or biopsy samples

• Validation in relevant disease contexts:

  • Test antibody performance in disease-relevant tissues

  • Validate against samples with altered CycH;1 expression

  • Compare results with established clinical biomarkers

• Quantification standardization:

  • Develop calibration standards for clinical sample analysis

  • Establish reference ranges for normal expression

  • Create SOPs for clinical laboratory implementation

• Ethical and regulatory considerations:

  • Obtain appropriate IRB approval for clinical sample testing

  • Document validation data for potential diagnostic applications

  • Consider regulatory requirements for clinical assay development