CYCU4-1 Antibody

What is CYCU4-1 and what biological systems does it function in?

CYCU4-1 (also listed as CYCP4;1) is a plant-specific cyclin protein involved in cell cycle regulation. The antibody specifically targets this protein in plant systems, with confirmed reactivity against Arabidopsis thaliana . CYCP4;1 belongs to the cyclin family, which regulates cell cycle progression by activating cyclin-dependent kinases (CDKs).

In plants, cyclins like CYCP4;1 are critical for controlling cell division, growth, and developmental processes. While mammalian cyclins such as CDK4 form complexes with D-type cyclins to drive G1-to-S phase progression , plant cyclins have evolved specialized functions. The CYCU4-1 Antibody provides researchers with a tool to detect and study this protein's expression and function throughout plant development and in response to environmental stimuli.

How does the specificity of CYCU4-1 Antibody compare to other cyclin antibodies?

CYCU4-1 Antibody differs significantly from antibodies targeting mammalian cyclins in several important ways:

• Species specificity: CYCU4-1 Antibody is specifically designed for plant research with reactivity against plant tissues , unlike antibodies such as those for Cyclin E1 which target human systems .

• Target protein: The antibody recognizes CYCP4;1 (Entrez Gene ID: 819082) , a plant-specific cyclin with unique structural and functional properties different from mammalian cyclins like CDK4 .

• Immunogen characteristics: CYCU4-1 Antibody is raised against recombinant Arabidopsis thaliana CYCP4;1 protein , ensuring specificity for plant research applications.

• Application optimization: While the antibody shares common applications like Western blotting with other cyclin antibodies, the optimal conditions are specifically tailored for plant tissue processing and protein extraction protocols.

Research has shown that antibody specificity is crucial for cyclin protein detection, as cyclins often exist in multiple isoforms that can show complex expression patterns similar to CDK4's multiple protein variants .

What experimental validation steps are necessary before using CYCU4-1 Antibody?

Before conducting experiments with CYCU4-1 Antibody, researchers should implement these validation steps:

• Specificity testing: Verify antibody specificity using the provided pre-immune serum as a negative control and the supplied antigen (200μg) as a positive control .

• Concentration optimization: Determine optimal antibody dilution through titration experiments for Western blot and ELISA applications. Start with recommended dilutions and adjust based on signal-to-noise ratio.

• Sample preparation verification: Confirm effective protein extraction from plant tissues using specialized plant extraction buffers that address the challenges of plant-specific compounds like polyphenols.

• Cross-reactivity assessment: If working with plant species other than Arabidopsis thaliana, perform cross-reactivity tests to ensure the antibody recognizes the CYCP4;1 homolog in your species of interest.

• Control inclusion planning: Design experiments to include appropriate controls, such as tissue samples with known high and low CYCP4;1 expression levels.

These validation steps parallel approaches used for other cyclin antibodies, where researchers have identified multiple protein isoforms that respond differently to various antibodies .

What are the optimal conditions for using CYCU4-1 Antibody in Western Blot experiments?

For optimal Western Blot results with CYCU4-1 Antibody, researchers should consider the following protocol guidelines:

• Sample preparation:

  • Extract proteins using plant-specific extraction buffers containing protease inhibitors

  • Include reducing agents to ensure proper protein denaturation

  • Quantify protein concentration using Bradford or BCA assay

  • Load 20-50μg total protein per lane, depending on CYCP4;1 abundance

• Gel electrophoresis:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Include molecular weight markers spanning 10-70 kDa range

  • Run at 100-120V to ensure proper protein resolution

• Transfer and blocking:

  • Transfer to PVDF membranes (preferred for plant proteins)

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

  • Include the pre-immune serum as a negative control

• Antibody incubation:

  • Dilute CYCU4-1 Antibody in blocking buffer (starting dilution 1:1000)

  • Incubate overnight at 4°C with gentle rocking

  • Perform at least four 10-minute TBST washes

• Detection:

  • Use HRP-conjugated anti-rabbit IgG secondary antibody (1:5000)

  • Develop using enhanced chemiluminescence

  • Validate specific bands using the supplied antigen control

This approach addresses the challenges of detecting cyclin proteins, which frequently appear as multiple bands or isoforms as observed with CDK4 in mammalian systems .

How should researchers optimize CYCU4-1 Antibody for ELISA applications?

For effective ELISA with CYCU4-1 Antibody:

• Plate preparation and coating:

  • Coat high-binding ELISA plates with plant protein extract (2-5μg/well)

  • Alternatively, use purified recombinant CYCP4;1 for standard curve generation

  • Coat overnight at 4°C in carbonate-bicarbonate buffer (pH 9.6)

• Blocking and antibody application:

  • Block with 3-5% BSA in PBS for 1-2 hours at room temperature

  • Apply CYCU4-1 Antibody at optimized dilution (typically start at 1:500-1:2000)

  • Incubate for 2 hours at room temperature or overnight at 4°C

• Controls integration:

  • Include wells with pre-immune serum at matching dilution as negative control

  • Use the supplied antigen for positive control and standard curve development

  • Run blank wells (no primary antibody) to assess secondary antibody background

• Signal development and quantification:

  • Use HRP-conjugated anti-rabbit secondary antibody

  • Develop with TMB substrate and measure absorbance at 450nm

  • Generate standard curves using serial dilutions of the antigen

• Data analysis:

  • Subtract background values from all readings

  • Calculate CYCP4;1 concentration using the standard curve

  • Normalize to total protein concentration for comparative analysis

This methodology ensures reliable quantification of CYCP4;1 in plant samples while accounting for the potential presence of multiple cyclin isoforms, similar to the protein multiplicity observed with CDK4 .

What controls are essential when investigating CYCP4;1 expression across developmental stages?

When studying CYCP4;1 expression throughout plant development using CYCU4-1 Antibody, these controls are critical:

• Experimental controls:

  • Include the pre-immune serum as a negative control to assess non-specific binding

  • Use the supplied antigen as a positive control to confirm antibody functionality

  • Apply multiple protein extraction methods to ensure complete recovery

• Developmental reference controls:

  • Include tissues with known high CYCP4;1 expression (e.g., meristematic regions)

  • Sample non-dividing tissues as low-expression controls

  • Process all developmental stages simultaneously to minimize technical variation

• Normalization controls:

  • Measure housekeeping proteins (e.g., actin, tubulin) as loading controls

  • Quantify total protein using stain-free technology or Ponceau staining

  • Consider using nuclear proteins as references when studying nuclear-localized cyclins

• Validation controls:

  • Confirm protein expression changes with transcript analysis (RT-qPCR)

  • Use immunostaining to verify tissue-specific expression patterns

  • When available, include CYCP4;1 mutant or overexpression lines

• Technical controls:

  • Process biological replicates from independent plants

  • Include technical replicates to assess procedural variation

  • Perform replicate experiments across different growth cycles

This comprehensive control strategy helps distinguish genuine developmental regulation from technical artifacts, similar to approaches used when studying complex expression patterns of cyclins like CDK4 .

How can CYCU4-1 Antibody be used to investigate cell cycle-dependent protein interactions?

CYCU4-1 Antibody enables sophisticated analysis of CYCP4;1 protein interactions throughout the cell cycle:

• Co-immunoprecipitation approaches:

  • Use CYCU4-1 Antibody to immunoprecipitate native CYCP4;1 complexes

  • Identify interaction partners through Western blot or mass spectrometry

  • Compare interaction profiles across cell cycle phases

  • Validate key interactions using reverse co-IP with antibodies against candidate partners

• Synchronization strategies:

  • Synchronize plant cell cultures using aphidicolin or hydroxyurea

  • Collect samples at defined cell cycle phases

  • Use CYCU4-1 Antibody to track CYCP4;1 levels and binding partners across the cycle

  • Compare with known cell cycle markers to correlate with specific phases

• In situ interaction analysis:

  • Apply proximity ligation assay (PLA) using CYCU4-1 Antibody and antibodies against suspected interaction partners

  • Visualize interactions in fixed plant tissues or cells

  • Correlate interaction signals with cell cycle markers

• Chromatin association studies:

  • Perform chromatin immunoprecipitation (ChIP) using CYCU4-1 Antibody

  • Identify genomic regions where CYCP4;1-containing complexes bind

  • Compare binding profiles across cell cycle stages

This approach provides insights into CYCP4;1 function comparable to studies of mammalian cyclins, where researchers have identified complex regulation of CDK4 at different cell cycle phases beyond the canonical G1 progression .

What techniques can overcome challenges in detecting low-abundance CYCP4;1 protein?

For detecting low-abundance CYCP4;1 in plant samples:

• Enhanced extraction techniques:

  • Use specialized plant protein extraction buffers containing PVPP, DTT, and protease inhibitor cocktails

  • Implement TCA/acetone precipitation to concentrate proteins and remove interfering compounds

  • Consider subcellular fractionation to enrich nuclear proteins where cyclins often localize

• Signal amplification methods:

  • Apply two-step detection with biotin-conjugated secondary antibody followed by streptavidin-HRP

  • Use high-sensitivity ECL substrates developed for low-abundance proteins

  • Consider tyramide signal amplification (TSA) for immunohistochemistry

• Sample enrichment strategies:

  • Perform immunoprecipitation with CYCU4-1 Antibody before Western blotting

  • Use size-exclusion or ion-exchange chromatography to fractionate and concentrate samples

  • Apply protein concentration methods specific for low-abundance plant proteins

• Specialized detection approaches:

  • Implement sandwich ELISA using capture and detection antibodies

  • Consider digital ELISA platforms (e.g., Single Molecule Array technology)

  • Explore highly-sensitive proximity extension assays for protein detection

These approaches address challenges similar to those encountered when studying multiple CDK4 isoforms, where researchers have needed to distinguish between closely related proteins present at different abundance levels .

How does phosphorylation state affect CYCP4;1 detection by CYCU4-1 Antibody?

Phosphorylation can significantly impact CYCP4;1 detection:

• Phosphorylation effects on antibody recognition:

  • Phosphorylation may alter epitope accessibility or antibody binding affinity

  • Multiple bands observed in Western blot may represent different phosphorylation states

  • De-phosphorylation treatment can change the abundance and migration pattern of detected proteins, similar to effects observed with CDK4

• Analytical approaches for phosphorylation assessment:

  • Compare untreated samples with those treated with lambda phosphatase

  • Use Phos-tag™ SDS-PAGE to separate phosphorylated from non-phosphorylated forms

  • Apply 2D gel electrophoresis to resolve different phospho-isoforms

• Methodological considerations:

  • Include phosphatase inhibitors in extraction buffers to preserve in vivo phosphorylation state

  • Use phospho-specific antibodies (if available) alongside CYCU4-1 Antibody

  • Consider phospho-proteomics to identify specific phosphorylation sites

• Interpretation guidelines:

  • Multiple bands may indicate phosphorylation rather than non-specific binding

  • Shifts in apparent molecular weight may reflect phosphorylation status

  • Changes in band intensity after phosphatase treatment confirm phosphorylation

Research on CDK4 has demonstrated that phosphorylation can affect protein mobility on SDS-PAGE and change antibody recognition patterns, with different phosphorylation states potentially representing functional variations of the protein .

What strategies can resolve multiple band patterns when using CYCU4-1 Antibody?

When encountering multiple bands with CYCU4-1 Antibody:

• Isoform and modification analysis:

  • Treat samples with phosphatase to determine if bands represent phospho-isoforms

  • Compare with known molecular weights of alternative splice variants

  • Use epitope mapping to determine if all bands share the antibody recognition site

  • Consider that multiple bands may represent genuine biological variants, as observed with CDK4

• Specificity confirmation techniques:

  • Perform peptide competition assay using the supplied antigen

  • Compare patterns between the pre-immune serum and CYCU4-1 Antibody

  • Include samples from different tissues to assess expression patterns

  • When available, include CYCP4;1 mutant tissue as a negative control

• Technical optimization approaches:

  • Adjust antibody concentration to improve specificity

  • Optimize blocking conditions to reduce non-specific binding

  • Modify washing stringency to eliminate weak cross-reactivities

  • Test different protein extraction protocols to preserve protein integrity

• Advanced analysis methods:

  • Use immunoprecipitation followed by mass spectrometry to identify each band

  • Apply 2D gel electrophoresis to separate proteins by both isoelectric point and molecular weight

  • Consider RNA-sequencing to identify potential splice variants

Research on cyclins has shown that multiple protein isoforms often exist, as exemplified by CDK4 which appears as multiple bands on Western blots that respond differently to various antibodies .

How can researchers distinguish between specific and non-specific signals?

To differentiate specific CYCP4;1 signals from non-specific background:

• Control-based validation:

  • Compare signals between CYCU4-1 Antibody and pre-immune serum under identical conditions

  • Perform peptide competition assays using the supplied antigen to confirm specificity

  • Include biological samples with known differential expression of CYCP4;1

• Technical validation approaches:

  • Test multiple antibody dilutions to identify concentrations that maximize specific signal while minimizing background

  • Compare different blocking agents (BSA, milk, commercial blockers) to reduce non-specific binding

  • Increase washing stringency (higher salt concentration, longer washing times)

• Cross-validation strategies:

  • Confirm results using alternative detection methods (e.g., mass spectrometry)

  • Compare protein detection results with mRNA expression data

  • When possible, use multiple antibodies targeting different epitopes

• Signal analysis techniques:

  • Quantify signal-to-noise ratio across different experimental conditions

  • Apply digital image analysis to objectively assess band specificity

  • Consider statistical approaches to distinguish significant signals from background

Research on cyclin proteins has shown that antibody specificity can vary significantly, with some antibodies detecting multiple protein isoforms while others show higher specificity for particular variants .

What approaches can resolve contradictions between Western blot and ELISA results?

When Western blot and ELISA results using CYCU4-1 Antibody show discrepancies:

• Technical comparison analysis:

  • Evaluate whether both methods used identical sample preparation procedures

  • Compare antibody concentrations and incubation conditions between methods

  • Assess whether the same control samples show consistent results across techniques

• Method-specific considerations:

  • Western blot detects denatured proteins while ELISA typically uses native proteins

  • Epitope accessibility may differ between methods

  • ELISA measures total protein concentration while Western blot separates by size

• Sample preparation influences:

  • Test whether different extraction buffers affect results in each method

  • Consider how protein denaturation might impact epitope recognition

  • Evaluate whether sample processing affects certain protein isoforms differently

• Scientific approaches to resolve contradictions:

  • Use a third method (e.g., immunoprecipitation, mass spectrometry) for validation

  • Perform spike-in recovery experiments to assess matrix effects in ELISA

  • Test serial dilutions of samples to identify potential concentration-dependent effects

• Data interpretation strategies:

  • Consider that Western blot may detect multiple isoforms separately while ELISA measures total signal

  • Evaluate whether contradictions relate to specific sample types or experimental conditions

  • Create a standardized protocol that yields consistent results across methods

These discrepancies are common when studying proteins with multiple isoforms, as seen with CDK4 where different antibodies detect distinct protein variants with varying efficiency .

How can researchers combine CYCU4-1 Antibody data with transcriptomics for pathway analysis?

Integrating CYCU4-1 Antibody protein detection with transcriptomic data:

• Experimental design for multi-omics integration:

  • Collect matched samples for both protein and RNA analysis

  • Include time-course sampling to capture dynamics and temporal relationships

  • Design experiments with sufficient biological replicates for statistical power

• Correlation analysis methods:

  • Compare CYCP4;1 protein levels (detected by CYCU4-1 Antibody) with mRNA expression

  • Calculate Pearson or Spearman correlation coefficients

  • Identify genes whose expression profiles correlate with CYCP4;1 protein levels

  • Look for discordant patterns that may indicate post-transcriptional regulation

• Pathway mapping strategies:

  • Use gene set enrichment analysis (GSEA) to identify pathways enriched among CYCP4;1-correlated genes

  • Apply network analysis to position CYCP4;1 within regulatory frameworks

  • Identify transcription factors that may regulate both CYCP4;1 and co-expressed genes

• Causal relationship investigation:

  • Design perturbation experiments (e.g., CYCP4;1 overexpression or knockdown)

  • Measure resulting changes in both transcriptome and proteome

  • Use statistical causal inference methods to infer regulatory relationships

This integration approach can reveal relationships between CYCP4;1 and other cell cycle regulators, similar to studies that have examined CDK4's complex regulation and its multiple potential isoforms .

What techniques complement CYCU4-1 Antibody detection for studying plant cell cycle regulation?

Complementary approaches to enhance CYCP4;1 research:

• Genetic and molecular tools:

  • Generate tagged CYCP4;1 lines (GFP, RFP, HA) for localization and interaction studies

  • Develop CRISPR/Cas9 knockouts or RNAi lines to assess loss-of-function phenotypes

  • Create overexpression lines to study gain-of-function effects

  • Apply promoter-reporter constructs to visualize transcriptional regulation

• Cell biology techniques:

  • Use flow cytometry to quantify cell cycle phases in wild-type versus CYCP4;1 mutants

  • Apply EdU labeling to measure S-phase progression

  • Implement live cell imaging to track division dynamics

  • Utilize fluorescence microscopy to monitor CYCP4;1 localization during cell cycle

• Biochemical approaches:

  • Perform in vitro kinase assays to assess CYCP4;1-CDK activity

  • Use size exclusion chromatography to study complex formation

  • Apply chromatin immunoprecipitation to identify genomic targets

  • Implement phosphoproteomics to identify substrates of CYCP4;1-associated kinases

• Systems biology integration:

  • Develop mathematical models of cell cycle regulation incorporating CYCP4;1

  • Apply network inference algorithms to position CYCP4;1 in regulatory networks

  • Use multi-omics data integration to comprehensively map cell cycle control

These complementary approaches can help understand the complex functions of plant cyclins, paralleling studies of mammalian cyclins like CDK4 that have revealed previously unrecognized functions at different cell cycle phases .

How should researchers design experiments to study CYCP4;1 post-translational modifications?

For investigating CYCP4;1 post-translational modifications:

• Detection strategies:

  • Use CYCU4-1 Antibody for immunoprecipitation followed by modification-specific detection

  • Apply Phos-tag™ gels to separate phosphorylated from non-phosphorylated forms

  • Compare band patterns before and after phosphatase treatment

  • Consider that multiple bands detected by antibodies may represent different modification states, as observed with CDK4

• Mass spectrometry approaches:

  • Immunoprecipitate CYCP4;1 using CYCU4-1 Antibody

  • Analyze by LC-MS/MS to identify specific modification sites

  • Implement targeted MS methods to quantify modification stoichiometry

  • Compare modification profiles across developmental stages or stress conditions

• Functional analysis:

  • Generate phospho-mimetic and phospho-null mutants of key residues

  • Express modified variants in plants to assess functional consequences

  • Measure effects on protein stability, localization, and interaction partners

  • Compare cell cycle progression in plants expressing different variants

• Experimental design considerations:

  • Include synchronization to capture cell cycle-specific modifications

  • Apply stress treatments to identify condition-dependent modifications

  • Design time-course experiments to track modification dynamics

  • Compare modifications across different tissues and developmental stages

Understanding CYCP4;1 post-translational modifications can provide insights into regulatory mechanisms, similar to studies showing how phosphorylation affects CDK4 mobility on SDS-PAGE and potentially its function .