CYCU3-1 Antibody

What is CYCU3-1 and what biological function does it serve?

CYCU3-1 is a plant-specific cyclin protein found in Arabidopsis thaliana (mouse-ear cress), a model organism in plant molecular biology. It belongs to the U-type cyclin family that functions as a regulatory component in the cell cycle progression. These cyclins associate with cyclin-dependent kinases (CDKs) to regulate various cellular processes including DNA replication and cell division in plants. U-type cyclins are particularly interesting as they are plant-specific and play specialized roles in plant growth and development. Understanding CYCU3-1 function contributes to broader knowledge of plant-specific cell cycle regulation mechanisms that differ from those in animal systems .

What applications is the CYCU3-1 antibody validated for?

The CYCU3-1 antibody has been specifically validated for ELISA (Enzyme-Linked Immunosorbent Assay) and Western Blotting (WB) applications using Arabidopsis thaliana samples. These applications allow researchers to detect and quantify the CYCU3-1 protein in complex biological samples. The antibody undergoes antigen affinity purification to ensure specific binding to the target protein, minimizing cross-reactivity with other cellular components. While the primary validated applications remain ELISA and WB, researchers might explore its utility in other immunological techniques after proper validation, though additional optimization may be required for techniques beyond the validated applications .

How should the CYCU3-1 antibody be stored to maintain optimal activity?

For optimal preservation of antibody activity, the CYCU3-1 antibody should be stored at either -20°C or -80°C immediately upon receipt. Repeated freeze-thaw cycles should be strictly avoided as they can lead to protein denaturation and loss of antibody binding capacity. The antibody is supplied in a storage buffer containing 0.03% Proclin 300 as a preservative and 50% glycerol in 0.01M PBS at pH 7.4, which helps maintain stability during freezing. For working solutions, small aliquots should be prepared to minimize the need for repeated freezing and thawing of the original stock. When handling the antibody, it's recommended to keep it on ice during experimental procedures to maintain its structural integrity and binding efficiency .

What is the species reactivity profile of the CYCU3-1 antibody?

The CYCU3-1 antibody has been specifically developed and validated for reactivity with Arabidopsis thaliana proteins. It targets the Q8LB60 UniProt protein (CYCU3-1) from this model plant organism. The antibody has not been validated for cross-reactivity with other plant species, including important crops or other model plant systems. This species-specific reactivity makes it particularly valuable for studies focused on Arabidopsis as a model system but requires careful consideration when extrapolating findings to other plant species. If researchers need to study CYCU3-1 homologs in other species, they would need to perform preliminary validation experiments to determine potential cross-reactivity based on sequence homology between the target proteins .

How can I optimize the CYCU3-1 antibody for cyclic immunofluorescence (cyCIF) applications?

While the CYCU3-1 antibody is not explicitly validated for immunofluorescence applications, researchers interested in adapting it for cyclic immunofluorescence should consider several optimization steps. First, perform a preliminary titration experiment with concentration gradients (typically ranging from 1:100 to 1:2000) to determine optimal antibody dilution for your specific sample type. For cyCIF applications specifically, the antibody can be conjugated with oligonucleotide barcodes using DNA-antibody conjugation kits following approaches similar to those described for other antibodies in multiplexed imaging.

Based on recent advances in flexible cyCIF using oligonucleotide conjugation, consider implementing signal amplification strategies to enhance detection sensitivity. This is particularly important for plant proteins that may be expressed at lower levels. The amplification can be achieved by using secondary antibodies conjugated with oligonucleotides that carry multiple fluorophore binding sites. This approach has shown increased signal-to-background ratios in similar applications. For tissue preservation during multiple rounds of imaging, use gentle UV treatment for fluorophore quenching between cycles rather than harsh stripping buffers that might damage delicate plant tissues .

How do different fixation protocols affect CYCU3-1 epitope accessibility in plant tissues?

The epitope accessibility of CYCU3-1 in plant tissues can be significantly affected by the fixation method employed. Plant tissues present unique challenges due to their cell walls and vacuoles that can impede antibody penetration. For CYCU3-1 detection, compare the following fixation protocols to determine optimal epitope preservation:

For optimal results with CYCU3-1, implement a controlled permeabilization step after fixation using a plant-specific enzymatic cocktail (e.g., cellulase, macerozyme, pectolyase) to improve antibody access to intracellular antigens without compromising epitope integrity. Additionally, antigen retrieval methods using citrate buffer (pH 6.0) at controlled temperatures may significantly enhance signal detection, particularly when working with paraffin-embedded plant tissues .

What approaches can mitigate cross-reactivity when studying CYCU3-1 in transgenic Arabidopsis lines?

When studying CYCU3-1 in transgenic Arabidopsis lines, particularly those expressing tagged versions of cyclins or in knock-in/knockout experiments, specific strategies can minimize cross-reactivity issues. First, implement rigorous negative controls including: (1) samples from cycu3-1 knockout lines to confirm antibody specificity, (2) pre-absorption controls where the antibody is pre-incubated with excess purified antigen before staining, and (3) isotype controls using non-specific rabbit IgG at matching concentrations.

For transgenic lines expressing tagged CYCU3-1 variants, consider a dual-detection approach comparing signals from both the CYCU3-1 antibody and an antibody against the tag (e.g., GFP, FLAG, or HA). Colocalization of signals provides stronger evidence of specificity. When analyzing multiple cyclin family members, which often share sequence homology, implement a competitive binding assay where recombinant proteins from related cyclins are used to determine potential cross-reactivity profiles.

Additionally, consider using computational modeling approaches similar to those employed in antibody specificity inference studies to predict potential cross-reactive epitopes based on sequence alignment with other plant cyclins. This bioinformatics-guided approach can help identify potential false positives before experimental validation .

How can I quantitatively analyze CYCU3-1 expression across different cell cycle phases?

For quantitative analysis of CYCU3-1 expression across different cell cycle phases in plant cells, implement a multiparametric approach combining antibody detection with cell cycle markers. Begin by synchronizing Arabidopsis cell cultures using aphidicolin (G1/S boundary), hydroxyurea (early S-phase), or propyzamide (M-phase) treatments followed by release and time-course sampling.

Establish a flow cytometry protocol using the CYCU3-1 antibody in combination with DNA content staining (using propidium iodide or DAPI) to correlate protein levels with specific cell cycle phases. For microscopy-based quantification, implement the following workflow:

• Co-stain samples with the CYCU3-1 antibody and antibodies against established cell cycle phase markers (e.g., PCNA for S-phase, phospho-histone H3 for M-phase)

• Acquire Z-stack confocal images with consistent exposure settings

• Perform quantitative image analysis using software like ImageJ/Fiji with specific macros for nuclear signal intensity measurement

• Normalize CYCU3-1 signal intensity to the internal control markers

• Plot expression profiles across the identified cell cycle phases

For Western blot quantification, implement a ratiometric approach comparing CYCU3-1 levels to multiple reference proteins to account for loading variations across synchronized samples. Quantitative RT-PCR can complement protein analysis by measuring transcript levels, though post-transcriptional regulation should be considered when interpreting results .

What is the optimal protocol for Western blotting using the CYCU3-1 antibody?

For optimal Western blotting using the CYCU3-1 antibody, employ the following specialized protocol developed for plant cyclin proteins:

Sample Preparation:

• Extract total proteins from Arabidopsis tissues using a plant-specific extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1 mM EDTA) supplemented with protease inhibitors and phosphatase inhibitors

• Include 1 mM DTT and 1 mM PMSF fresh before extraction

• Clarify lysates by centrifugation at 14,000 × g for 15 minutes at 4°C

• Quantify protein using Bradford or BCA assay

Gel Electrophoresis and Transfer:

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

• Include a positive control (recombinant CYCU3-1 protein if available)

• Transfer proteins to PVDF membrane at 25V constant for 2 hours in cold transfer buffer containing 10% methanol

Antibody Incubation:

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

• Incubate with CYCU3-1 antibody at 1:500 to 1:1000 dilution in 2% BSA in TBST overnight at 4°C

• Wash 4 times (10 minutes each) with TBST

• Incubate with HRP-conjugated anti-rabbit secondary antibody at 1:5000 dilution for 1 hour at room temperature

• Wash 4 times (10 minutes each) with TBST

Detection:

• Apply ECL substrate and expose to X-ray film or capture using a digital imaging system

• Expected molecular weight for CYCU3-1 is approximately 45-50 kDa

For quantitative analysis, include an internal loading control such as anti-actin or anti-GAPDH antibodies and perform densitometric analysis using ImageJ or similar software. If signal-to-noise ratio is insufficient, consider using a more sensitive detection method such as enhanced chemiluminescence Plus (ECL Plus) or fluorescently-labeled secondary antibodies for digital imaging systems .

How should I prepare plant tissue samples for ELISA using the CYCU3-1 antibody?

For ELISA applications using the CYCU3-1 antibody with plant tissue samples, the following optimized protocol addresses the unique challenges of plant protein extraction and detection:

Sample Preparation:

• Collect fresh Arabidopsis tissue samples (preferably young leaves or seedlings where cell division is active)

• Flash-freeze samples in liquid nitrogen and grind to a fine powder using a pre-chilled mortar and pestle

• Extract proteins using a specialized plant protein extraction buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mM EDTA, 2 mM DTT)

• Add plant-specific protease inhibitor cocktail with additional 1 mM PMSF, 10 mM sodium fluoride, and 1 mM sodium orthovanadate

• Centrifuge at 15,000 × g for 20 minutes at 4°C

• Collect supernatant and determine protein concentration

ELISA Protocol:

• Coat high-binding 96-well ELISA plates with 50-100 μl of protein extract (10-50 μg/ml in carbonate-bicarbonate buffer, pH 9.6) or coating antibody (for sandwich ELISA)

• Incubate overnight at 4°C

• Wash 3 times with PBST (PBS + 0.05% Tween-20)

• Block with 3% BSA in PBST for 2 hours at room temperature

• For direct ELISA: Add CYCU3-1 antibody at 1:500-1:2000 dilution in 1% BSA/PBST
  For sandwich ELISA: Add sample diluted in 1% BSA/PBST, followed by CYCU3-1 antibody

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

• Wash 5 times with PBST

• Add HRP-conjugated secondary antibody at 1:5000 dilution in 1% BSA/PBST

• Incubate for 1 hour at room temperature

• Wash 5 times with PBST

• Add TMB substrate and incubate for 15-30 minutes

• Stop reaction with 2N H₂SO₄

• Read absorbance at 450 nm

For quantitative analysis, prepare a standard curve using recombinant CYCU3-1 protein if available. Include negative controls using extract from cycu3-1 knockout plants or pre-immune serum to determine background signal levels .

What controls should be included when validating CYCU3-1 antibody specificity in my experimental system?

When validating the specificity of the CYCU3-1 antibody in your experimental system, implement a comprehensive set of controls to ensure reliable and interpretable results:

Essential Primary Controls:

• Genetic Controls: Include samples from cycu3-1 knockout/knockdown lines, which should show significantly reduced or absent signal

• Antigen Competition: Pre-incubate the antibody with excess purified recombinant CYCU3-1 protein before sample application; specific binding should be blocked

• Isotype Control: Use non-specific rabbit IgG at the same concentration to identify non-specific binding

• Cross-Reactivity Assessment: Test the antibody against recombinant proteins of other cyclin family members with sequence similarity

Secondary Validation Controls:

• Molecular Weight Verification: Confirm that the detected band in Western blots corresponds to the expected molecular weight of CYCU3-1 (approximately 45-50 kDa)

• Alternative Antibody Comparison: If available, compare results with another antibody targeting a different epitope of CYCU3-1

• Expression Pattern Correlation: Compare protein detection with known mRNA expression patterns from published RNA-seq or qRT-PCR data

• Subcellular Localization: Verify that the detected protein localizes to the expected cellular compartments based on CYCU3-1's known biology

Advanced Validation Approaches:

Document all validation experiments methodically, including positive and negative results, to establish a comprehensive specificity profile for the CYCU3-1 antibody in your specific experimental conditions .

What are common issues when using CYCU3-1 antibody in Western blots and how can they be resolved?

When working with the CYCU3-1 antibody in Western blotting applications, researchers may encounter several common issues. Here are problem-solving approaches for each:

Issue: Weak or No Signal

• Cause: Insufficient protein, antibody concentration too low, protein degradation

• Solution:

  • Increase protein loading to 50-75 μg

  • Increase primary antibody concentration (try 1:250 dilution)

  • Extend primary antibody incubation to overnight at 4°C

  • Enhance extraction buffer with additional protease inhibitors

  • Use freshly prepared samples and avoid repeated freeze-thaw cycles

Issue: Multiple Bands/Non-specific Binding

• Cause: Cross-reactivity with related cyclins, sample degradation, secondary antibody issues

• Solution:

  • Increase blocking time/concentration (try 5% BSA instead of milk)

  • Dilute antibody in 2% BSA with 0.05% sodium azide

  • Increase wash stringency (add 0.1% SDS to TBST wash buffer)

  • Pre-absorb antibody with total protein extract from cycu3-1 knockout plants

  • Reduce secondary antibody concentration

Issue: High Background

• Cause: Insufficient blocking, excessive antibody, membrane contamination

• Solution:

  • Extend blocking to 2-3 hours at room temperature

  • Add 0.1-0.2% Tween-20 to antibody dilution buffer

  • Increase number and duration of washes (6 x 10 minutes)

  • Filter all solutions to remove particulates

  • Use fresh transfer buffer and high-quality methanol

Issue: Inconsistent Results Between Experiments

• Cause: Variable extraction efficiency, inconsistent transfer, unstable antibody

• Solution:

  • Standardize tissue collection (same growth stage, time of day, tissue type)

  • Include internal control for protein extraction efficiency

  • Use semi-dry transfer system with constant current parameters

  • Aliquot antibody upon first use to avoid repeated freeze-thaw

  • Validate each new antibody lot against a reference sample

For plant-specific samples, consider adding 2% polyvinylpyrrolidone (PVP) to extraction buffers to remove plant phenolic compounds that might interfere with protein detection. Additionally, for low abundance CYCU3-1 detection, incorporate a protein concentration step using methanol/chloroform precipitation before gel loading .

How can I improve signal sensitivity for detecting low-abundance CYCU3-1 protein?

Detecting low-abundance CYCU3-1, particularly in non-dividing or specialized plant tissues, requires enhanced sensitivity approaches. Implement these advanced techniques to improve detection limits:

Enhanced Sample Preparation:

• Implement subcellular fractionation to enrich for nuclear proteins, where CYCU3-1 is predominantly localized

• Use phosphatase inhibitor cocktails specifically designed for plant tissues (containing cantharidic acid, bromotetramisole, and microcystin-LR)

• Perform protein precipitation with TCA/acetone followed by resuspension in a smaller volume to concentrate proteins

Signal Amplification Strategies:

• Utilize tyramide signal amplification (TSA) system, which can increase sensitivity by 10-100 fold over conventional detection methods

• Implement a biotin-streptavidin detection system using biotinylated secondary antibodies followed by HRP-conjugated streptavidin

• Consider using amplification oligonucleotides attached to antibodies, similar to those used in cyclic immunofluorescence, which can significantly increase signal-to-background ratios

Alternative Detection Methods:

• Switch to chemiluminescent substrates with enhanced sensitivity (SuperSignal West Femto Maximum Sensitivity Substrate)

• Use fluorescently-labeled secondary antibodies with digital imaging systems that offer greater dynamic range

• Consider ultrasensitive ELISA formats like the proximity ligation assay (PLA) that can detect single protein molecules

Protocol Modifications:

• Extend primary antibody incubation time to 36-48 hours at 4°C with gentle agitation

• Use polymeric HRP-conjugated secondary antibody systems that provide multiple HRP molecules per binding event

• Add 5-10% dextran sulfate to primary antibody incubation buffer to enhance binding kinetics

For Western blotting specifically, consider using gradient gels (4-20%) to improve protein separation and transfer, and implement PVDF membranes with smaller pore size (0.2 μm) to prevent protein loss during transfer. When using imaging systems, increase exposure times incrementally while monitoring background to determine optimal signal-to-noise ratio .

What approaches can resolve epitope masking issues when detecting CYCU3-1 in fixed plant tissues?

Epitope masking is a common challenge when detecting CYCU3-1 in fixed plant tissues, particularly due to plant-specific factors like cell wall components and vacuoles. Implement these specialized approaches to improve epitope accessibility:

Optimized Fixation Protocols:

• Test a gradient of fixation times (10 minutes to 24 hours) to determine minimal fixation required for tissue preservation

• Compare cross-linking fixatives (4% paraformaldehyde) with precipitating fixatives (methanol/acetone mixtures) for epitope preservation

• Consider dual fixation approach: brief aldehyde fixation (15-20 minutes) followed by cold methanol/acetone (10 minutes) to preserve both structure and antigenicity

Antigen Retrieval Methods:

• Heat-induced epitope retrieval in citrate buffer (10 mM sodium citrate, pH 6.0) at 95°C for 10-20 minutes

• Enzymatic antigen retrieval using plant-specific enzyme cocktails (1 mg/ml pectolyase, 0.5 mg/ml cellulase, 0.1% Triton X-100) at 37°C for 15-30 minutes

• Microwave-assisted retrieval using alkaline buffer (Tris-EDTA, pH 9.0) with controlled temperature cycling

Detergent-Based Permeabilization Optimization:

• Sequential permeabilization: Start with mild detergent (0.1% Triton X-100) followed by stronger surfactant (0.5% Saponin)

• Implement freeze-thaw cycles (3-5 cycles of liquid nitrogen/37°C water bath) after mild fixation to create microscopic ice crystals that improve antibody penetration

• Test digitonin (25-50 μg/ml) for selective permeabilization of plasma membrane while preserving nuclear membrane integrity

Advanced Signal Visualization:

• Implement tyramide signal amplification (TSA) with DNP or biotin-labeled tyramides

• Use quantum dot-conjugated secondary antibodies for improved signal stability and brightness

• Consider multiplex immunofluorescence approaches that allow detection of CYCU3-1 alongside tissue-specific markers

For particularly challenging samples, implement a progressive antigen retrieval approach where you incrementally increase the intensity of retrieval conditions while monitoring tissue integrity and specific signal-to-noise ratio. Document optimal conditions for different tissue types, developmental stages, and experimental conditions to establish a comprehensive protocol for reliable CYCU3-1 detection .