CYCT1-1 Antibody

What is CYCT1-1 antibody and what is its target antigen?

CYCT1-1 antibody is an immunological reagent designed to detect and bind to cyclin T1, specifically the CYCT1-1 protein in plant samples. The target antigen is encoded by the CYCT1-1 gene, which is a plant homolog of the mammalian CCNT1 gene. In humans, cyclin T1 is a nuclear protein with 726 amino acid residues and a molecular weight of approximately 80.7 kilodaltons, with two identified isoforms . In plants, CYCT1-1 functions as part of the transcriptional machinery, though with structural and functional differences from its mammalian counterpart. The antibody enables researchers to study the expression, localization, and interactions of CYCT1-1 in various plant tissues and experimental conditions .

What are the primary applications of CYCT1-1 antibody in plant research?

CYCT1-1 antibody is primarily used for Western Blot (WB) and Enzyme-Linked Immunosorbent Assay (ELISA) applications in plant research . These applications allow researchers to:

• Quantify CYCT1-1 protein expression levels in different plant tissues, developmental stages, or treatment conditions

• Assess protein localization in cellular fractions

• Evaluate protein-protein interactions through co-immunoprecipitation experiments

• Monitor changes in CYCT1-1 expression during different physiological or stress conditions

• Validate genetic manipulation outcomes in transgenic plants

The antibody specifically recognizes plant CYCT1-1 proteins, making it suitable for studies in Arabidopsis and other plant species but not for animal or human samples .

How should CYCT1-1 antibody be stored and handled to maintain its activity?

For optimal performance and longevity of CYCT1-1 antibody:

• Store the antibody at -20°C for long-term storage

• Avoid repeated freeze-thaw cycles by preparing small aliquots upon receipt

• When working with the antibody, keep it on ice or at 4°C

• Dilute only the amount needed for immediate use in appropriate buffer

• Monitor storage conditions regularly to ensure temperature stability

• Check expiration dates and antibody clarity before use (cloudiness or precipitation may indicate deterioration)

• For reconstituted lyophilized antibodies, follow manufacturer's recommendations for buffer composition

Proper storage and handling are essential for maintaining antibody specificity and sensitivity in experimental applications.

What are the optimal protocols for using CYCT1-1 antibody in Western blotting of plant samples?

For successful Western blotting with CYCT1-1 antibody in plant samples:

Sample Preparation:

• Extract total protein from plant tissue using a plant-specific extraction buffer containing protease inhibitors

• Determine protein concentration using Bradford or BCA assay

• Prepare samples in Laemmli buffer with reducing agent and heat at 95°C for 5 minutes

Gel Electrophoresis and Transfer:

• Load 20-50 μg of protein per lane on a 10-12% SDS-PAGE gel

• Use prestained molecular weight markers to track migration

• Transfer to PVDF membrane (preferred over nitrocellulose for plant proteins)

• Verify transfer efficiency with reversible staining (Ponceau S)

Antibody Incubation:

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

• Incubate with CYCT1-1 antibody at 1:1000 dilution in blocking buffer overnight at 4°C

• Wash 4 times with TBST, 5 minutes each

• Incubate with secondary antibody (anti-mouse HRP) at 1:5000 dilution for 1 hour at room temperature

• Wash 4 times with TBST, 5 minutes each

Detection:

• Apply chemiluminescent substrate and capture signal using imaging system

• Expected molecular weight for Arabidopsis CYCT1-1 is approximately 55-60 kDa

• Validate specificity using positive and negative controls

This protocol may require optimization based on specific plant species and tissue types being analyzed .

How can researchers troubleshoot weak or non-specific signals when using CYCT1-1 antibody?

When encountering weak or non-specific signals with CYCT1-1 antibody, consider the following troubleshooting approaches:

For Weak Signals:

• Increase antibody concentration (try 1:500 instead of 1:1000)

• Extend primary antibody incubation time to 24-48 hours at 4°C

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

• Use enhanced sensitivity chemiluminescent substrates

• Optimize extraction buffer to ensure efficient protein extraction from plant tissues

• Consider using a detection system with signal amplification

For Non-specific Signals:

• Increase blocking stringency (5-10% blocking agent)

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

• Pre-absorb antibody with plant extract from negative control tissue

• Increase washing duration and number of washes

• Decrease secondary antibody concentration

• Use gradient gels to better separate proteins of similar molecular weights

• Consider using a monoclonal alternative if available

General Optimization:

• Test different membrane types (PVDF vs. nitrocellulose)

• Optimize transfer conditions for high molecular weight proteins

• Include appropriate positive and negative controls in each experiment

• Verify protein integrity during extraction using Coomassie-stained gels

Methodical troubleshooting and careful documentation of conditions will help identify the optimal parameters for specific experimental systems.

What controls should be included when using CYCT1-1 antibody in plant research experiments?

Rigorous experimental design requires appropriate controls when using CYCT1-1 antibody:

Essential Controls:

• Positive Control: Include protein extract from plants with known CYCT1-1 expression (e.g., Arabidopsis seedlings)

• Negative Control: Use one of the following:

  • Extract from cyct1-1 knockout/knockdown plants

  • Pre-immune serum in place of primary antibody

  • Secondary antibody only (omitting primary antibody)

• Loading Control: Probe the same membrane with antibodies against constitutively expressed proteins:

  • Actin (plant-specific anti-actin antibody)

  • GAPDH

  • Tubulin

• Peptide Competition: Pre-incubate antibody with excess CYCT1-1 peptide to confirm specificity

• Cross-Reactivity Control: Test antibody on protein extracts from non-target plant species

Technical Controls:

Including these controls helps validate results and troubleshoot experimental issues, ensuring reliable and reproducible findings .

How can CYCT1-1 antibody be used to investigate plant-pathogen interactions?

CYCT1-1 antibody can be employed in sophisticated experimental designs to elucidate the role of cyclin T1 in plant-pathogen interactions:

Infection Time Course Analysis:

• Infect plants with pathogens (viral, bacterial, or fungal)

• Collect samples at various timepoints post-infection

• Perform Western blot analysis with CYCT1-1 antibody to track changes in expression levels

• Correlate CYCT1-1 expression with disease progression or resistance

Subcellular Fractionation:

• Separate infected plant tissues into nuclear, cytoplasmic, and membrane fractions

• Analyze CYCT1-1 localization in each fraction using the antibody

• Determine if pathogen infection alters CYCT1-1 cellular distribution

Co-Immunoprecipitation (Co-IP):

• Prepare lysates from infected and control plants

• Use CYCT1-1 antibody for immunoprecipitation

• Analyze co-precipitating proteins by mass spectrometry

• Identify pathogen effectors or plant defense proteins that interact with CYCT1-1

Chromatin Immunoprecipitation (ChIP):

• Perform ChIP with CYCT1-1 antibody on infected vs. control plants

• Sequence precipitated DNA (ChIP-seq) to identify genomic regions bound by CYCT1-1

• Analyze if pathogen infection alters CYCT1-1 recruitment to defense-related genes

This multifaceted approach can reveal whether plant pathogens target or manipulate CYCT1-1 function during infection, potentially uncovering new mechanisms of plant immunity or pathogen virulence strategies.

What insights can be gained from studying CYCT1-1 in relation to HIV-1 Tat protein interactions?

While CYCT1-1 antibody specifically targets plant cyclin T1, comparative studies between plant and human cyclin T1 can provide valuable insights into HIV-1 Tat interactions:

Structural Comparison Studies:

• Use CYCT1-1 antibody to purify plant cyclin T1

• Compare structural features with human cyclin T1 using X-ray crystallography or cryo-EM

• Identify conserved and divergent regions that might explain species-specific interactions with Tat

Functional Domain Analysis:
Research has shown that human cyclin T1 contains a Tat-recognition motif (TRM) that is critical for HIV-1 Tat binding and transcriptional activation . Comparative studies can:

• Evaluate if plant CYCT1-1 contains similar structural elements

• Test chimeric constructs combining plant and human cyclin T1 domains

• Use CYCT1-1 antibody to verify expression and localization of these constructs

Therapeutic Development Insights:
Studies have identified compounds that inhibit HIV-1 replication by targeting the cyclin T1-Tat interaction . The study of plant CYCT1-1 may:

• Provide evolutionary context for structure-function relationships

• Identify naturally evolved resistance mechanisms in plant cyclins

• Inform design of more specific inhibitors targeting human cyclin T1

The molecular dynamics simulation studies of cyclin T1-Tat interactions have revealed that the dynamic structural change of cyclin T1 H2' helix is indispensable for its activity in Tat function . Understanding the structural parallels in plant CYCT1-1 could provide evolutionary insights into this important protein family.

How does the phosphorylation status of CYCT1-1 affect its function and detection by antibodies?

Post-translational modifications, particularly phosphorylation, can significantly impact CYCT1-1 function and detection:

Impact on Antibody Detection:

• Phosphorylation may alter epitope accessibility or antibody binding affinity

• Some antibody preparations may preferentially recognize phosphorylated or non-phosphorylated forms

• To comprehensively detect all forms of CYCT1-1:

  • Use multiple antibodies targeting different epitopes

  • Compare results from native and denaturing conditions

  • Consider using phosphorylation-state specific antibodies if available

Functional Analysis of Phosphorylation:

• Treat protein extracts with phosphatases before immunoblotting

• Compare migration patterns of treated vs. untreated samples

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

• Perform 2D gel electrophoresis to resolve different phosphorylated species

Experimental Approach to Study Phosphorylation:

In human cells, the phosphorylation of cyclin T1 affects its interaction with Tat and RNA polymerase II, which is crucial for HIV-1 transcription . Similar regulatory mechanisms may exist in plant CYCT1-1, though with different functional outcomes due to the absence of viral transcription factors like Tat in plants.

How does research on plant CYCT1-1 contribute to understanding human cyclin T1 in HIV-1 pathogenesis?

While studying plant CYCT1-1 with specific antibodies, researchers can gain comparative insights relevant to human HIV-1 pathogenesis:

Evolutionary Conservation Analysis:

• Use CYCT1-1 antibody to identify and purify plant cyclin T1

• Perform sequence and structural comparisons with human cyclin T1

• Identify functionally conserved domains that may be essential for transcriptional regulation

• Map species-specific differences that explain why plant cyclins cannot support HIV-1 transcription

Functional Domain Studies:
Molecular dynamics simulation and experimental verification have shown that the dynamic structural change of human cyclin T1 H2' helix is indispensable for its activity in Tat function . Comparative studies can:

• Analyze if plant CYCT1-1 contains similar structural elements

• Study the flexibility of corresponding regions in plant cyclin T1

• Identify naturally evolved structural variations that prevent Tat binding

Alternative Models for Therapeutic Development:

• Express human-plant chimeric cyclin T1 proteins in plant systems

• Use CYCT1-1 antibody to confirm expression and proper folding

• Test candidate HIV-1 transcription inhibitors against these constructs

• Leverage plant systems as preliminary screening platforms for drug discovery

This cross-kingdom comparative approach can identify fundamental principles of cyclin T1 function that might be leveraged for therapeutic development against HIV-1 .

What methodological approaches can be used to study CYCT1-1 interactions with plant-specific transcription factors?

To investigate interactions between CYCT1-1 and plant-specific transcription factors, researchers can employ several sophisticated approaches:

Co-Immunoprecipitation (Co-IP) and Pull-Down Assays:

• Use CYCT1-1 antibody for immunoprecipitation from plant extracts

• Identify co-precipitating proteins by mass spectrometry

• Validate interactions with candidate transcription factors using reciprocal Co-IP

• For transient interactions, consider using crosslinking before extraction

Bimolecular Fluorescence Complementation (BiFC):

• Generate fusion constructs of CYCT1-1 and candidate transcription factors with split fluorescent protein fragments

• Express in plant protoplasts or through transient transformation

• Analyze reconstituted fluorescence using confocal microscopy

• Include appropriate controls to verify specificity of interactions

Yeast Two-Hybrid (Y2H) and Split-Ubiquitin Systems:

• Create bait constructs with CYCT1-1 and prey constructs with candidate transcription factors

• Screen for interactions in yeast systems

• Validate positive interactions using deletion constructs to map interaction domains

• Confirm with in planta methods like Co-IP using CYCT1-1 antibody

Chromatin Immunoprecipitation followed by Sequencing (ChIP-seq):

• Perform ChIP with CYCT1-1 antibody

• Sequence precipitated DNA to identify genomic binding regions

• Compare with binding sites of candidate transcription factors

• Identify co-occupied regions suggesting functional interactions

These methodologies can reveal plant-specific transcription networks involving CYCT1-1, which may differ significantly from the well-characterized interactions of human cyclin T1 with transcription factors and the HIV-1 Tat protein .

How do different CYCT1-1 antibody preparations compare in terms of specificity and sensitivity?

When evaluating different CYCT1-1 antibody preparations for research applications, consider these comparative parameters:

Comparison of Antibody Types:

Performance Evaluation Methods:

• Side-by-side Western blot comparison using the same samples and conditions

• Epitope mapping to determine binding regions

• Cross-reactivity testing against related plant cyclins

• Evaluation in knockout/knockdown plant materials

Application-Specific Considerations:

• For Western blot: Evaluate background levels and signal-to-noise ratio

• For immunoprecipitation: Compare efficiency of target protein recovery

• For ChIP: Assess enrichment of known target sequences

• For ELISA: Compare detection limits and dynamic range

When selecting a CYCT1-1 antibody, researchers should consider running validation experiments with their specific plant species and experimental conditions to determine which preparation offers optimal performance for their research needs .

What are the emerging applications of CYCT1-1 antibody in plant stress response research?

CYCT1-1 antibody is increasingly being utilized in cutting-edge research on plant stress responses:

Stress-Induced Expression Profiling:

• Subject plants to various stresses (drought, salinity, pathogens, heat)

• Use CYCT1-1 antibody for Western blot analysis at different time points

• Correlate CYCT1-1 protein levels with transcriptional activity changes

• Identify stress conditions that specifically modulate CYCT1-1 expression or localization

Chromatin Dynamics During Stress:

• Perform ChIP-seq with CYCT1-1 antibody under normal and stress conditions

• Map genome-wide redistribution of CYCT1-1 during stress response

• Integrate with transcriptome data to correlate CYCT1-1 binding with gene expression changes

• Identify stress-specific transcriptional programs regulated by CYCT1-1

Post-Translational Modification Analysis:

• Use CYCT1-1 antibody for immunoprecipitation from stressed plant tissues

• Analyze precipitated proteins by mass spectrometry to identify stress-induced modifications

• Generate phospho-specific antibodies if key regulatory sites are identified

• Map kinase pathways that regulate CYCT1-1 during stress responses

Protein Complex Remodeling:

• Compare CYCT1-1 interaction partners under normal and stress conditions

• Identify stress-specific protein-protein interactions

• Determine if stress alters CYCT1-1 association with the transcriptional machinery

• Investigate if CYCT1-1 recruits specific stress-response transcription factors

These emerging applications highlight the importance of CYCT1-1 antibody as a tool for understanding plant adaptation mechanisms to environmental challenges.

What are the considerations for using CYCT1-1 antibody in cross-species studies?

When applying CYCT1-1 antibody across different plant species, researchers should address several important considerations:

Epitope Conservation Assessment:

• Perform sequence alignment of CYCT1-1 across target species

• Identify the epitope recognized by the antibody (contact supplier for this information)

• Evaluate conservation of this epitope region in target species

• Predict potential cross-reactivity based on sequence homology

Validation in Each Species:

• Begin with Western blot analysis to confirm detection and determine apparent molecular weight

• Verify specificity using genetic controls when available (knockouts, RNAi lines)

• Determine optimal working conditions, which may vary between species

• Consider using multiple antibodies targeting different epitopes for confirmation

Optimization Guidelines by Application:

Evolutionary Interpretation:

• Use phylogenetic analysis to contextualize differences in antibody reactivity

• Consider evolutionary distance when interpreting cross-reactivity patterns

• Leverage cross-species comparisons to identify conserved functional domains

• Document species-specific variations that may reflect adaptive evolution

Carefully validated cross-species applications of CYCT1-1 antibody can provide valuable comparative insights into cyclin T1 function across plant lineages .