At5g58770 Antibody

What is the At5g58770 gene and what protein does it encode?

At5g58770 is a gene locus in Arabidopsis thaliana that encodes AtCPT7 (Arabidopsis thaliana cis-Prenyltransferase 7), a member of the cis-prenyltransferase family. AtCPT7 plays a crucial role in polyprenol synthesis, specifically synthesizing medium-chain polyprenols of approximately 55 carbons in length. The enzyme uses geranylgeranyl diphosphate (GGPP) and isopentenyl diphosphate as substrates for this synthesis process .

Research with knockout mutants (cpt7−/−), RNAi lines, and overexpression lines has confirmed that AtCPT7 is essential for the production of medium-chain polyprenols in Arabidopsis leaves. Quantitative RT-PCR analysis has shown that AtCPT7 transcripts are virtually undetectable in T-DNA knockout lines and significantly reduced in RNAi lines, correlating with dramatically decreased polyprenol content .

Where is AtCPT7 protein localized in plant cells?

Experimental evidence from immunodetection and in vivo localization studies using AtCPT7 fluorescent protein fusions has definitively shown that AtCPT7 resides in the stroma of mesophyll chloroplasts . This chloroplast localization is directed by an N-terminal targeting sequence.

While the enzyme itself is stromal, its enzymatic products (polyprenols) accumulate in thylakoid membranes, where they play important roles in membrane stability and photosystem function. This dual localization pattern—stromal enzyme with membrane-embedded products—is a critical consideration when designing experiments to study AtCPT7 distribution and function .

What detection methods are available for studying AtCPT7 protein?

Several complementary techniques can be employed to detect and study AtCPT7:

• Immunoblot analysis using antibodies specific to AtCPT7

• Fluorescent protein fusions (e.g., GFP-AtCPT7) for in vivo localization

• Purification and detection of recombinant AtCPT7 expressed in E. coli

• Indirect detection through analysis of polyprenol content in different plant genotypes

The search results demonstrate that immunoblot analysis has been successfully used to assess the purity of cellular fractions when studying AtCPT7 localization . Additionally, recombinant AtCPT7 has been expressed in E. coli as a C-terminal fusion protein with a hexahistidine tag and purified using Ni²⁺-affinity chromatography for in vitro enzymatic studies .

What is the optimal sample preparation protocol for AtCPT7 immunodetection in different plant tissues?

For reliable detection of AtCPT7 in plant tissues, researchers should consider multiple methodological factors:

First, tissue selection is crucial. Research has shown that AtCPT7 is expressed in leaves but expression patterns differ from other CPT family members. For instance, while AtCPT2 (At2g23400) is absent in stem tissue, AtCPT7 is expressed in stems which contain polyprenols . This differential expression pattern should inform tissue selection for immunodetection experiments.

For subcellular fractionation, careful isolation procedures are necessary to preserve protein localization. When preparing chloroplast fractions, gentle isolation techniques should be used to maintain chloroplast integrity, followed by further fractionation into stromal and thylakoid membrane components. The purity of each fraction must be assessed using immunoblot analysis with specific antibodies against known compartment markers .

Due to the limited number of cells expressing specific proteins in specialized tissues (as noted in search result regarding QC cells), optimization of protein extraction methods may be required to enhance detection sensitivity. Complete protein extraction buffers containing appropriate detergents are essential, especially for membrane-associated proteins.

How can I validate the specificity of an At5g58770 antibody?

Validating antibody specificity is critical for reliable research results. Multiple complementary approaches should be employed:

• Genetic validation using knockout and overexpression lines:

  • Compare immunoblot signals between wild-type and cpt7−/− knockout mutants (e.g., SALK_022111 line)

  • Test RNAi lines with reduced AtCPT7 expression (e.g., RNAi-23, -24, and -31 lines)

  • Include AtCPT7 overexpression lines (CPT7-OE) which should show enhanced signal

  • Examine heterozygous plants from backcrosses (cpt7−/− × Col-0) which should show intermediate signal levels

• Protein-based validation methods:

  • Use purified recombinant AtCPT7 protein as a positive control

  • Perform epitope-blocking experiments to confirm binding specificity

  • Test cross-reactivity with other CPT family members that share sequence homology

Research demonstrates that AtCPT7 gene expression correlates with polyprenol content in various genetic backgrounds, providing an indirect validation method. For example, polyprenol content is significantly decreased in cpt7−/− and RNAi lines, while it is increased in overexpression lines .

What bioinformatic approaches can enhance antibody-based studies of AtCPT7?

Modern bioinformatic methods can significantly enhance antibody-based studies of AtCPT7:

• Epitope prediction and antibody design:

  • Computational analysis of protein structure to identify optimal epitopes

  • Prediction of antigenic regions with high specificity for AtCPT7 versus other CPT family members

  • Structure-based antibody design approaches as described in search result

• Data integration strategies:

  • Combining ChIP-chip, RNA-seq, CUT&RUN, and ATAC-seq data for comprehensive analysis

  • Search result describes approaches for "Data Integration" that can enhance understanding of protein function

  • Integration of transcriptomic, proteomic, and functional data to build comprehensive models

• Sequence analysis for cross-species applications:

  • Comparative sequence analysis to predict antibody cross-reactivity across plant species

  • Identification of conserved epitopes for developing antibodies with broader research applications

  • Machine learning approaches for predicting epitope conservation and antibody specificity

These bioinformatic approaches can provide valuable insights for experimental design, data interpretation, and development of more specific and effective antibodies for AtCPT7 research.

How should I design experiments to study AtCPT7's role in thylakoid membrane dynamics?

Research has demonstrated that AtCPT7's products (polyprenols) accumulate in thylakoid membranes and in their absence, "thylakoids adopt an increasingly 'fluid membrane' state" . Designing experiments to study this function requires careful consideration of several factors:

• Comprehensive phenotypic analysis:

  • Perform chlorophyll fluorescence measurements to assess photosystem II operating efficiency in wild-type versus AtCPT7-deficient plants

  • Measure electron transport rates in isolated thylakoids from different genotypes

  • Analyze thylakoid membrane fluidity using biophysical techniques (e.g., fluorescence anisotropy)

  • Correlate membrane dynamics with polyprenol content using lipidomic approaches

• Subcellular localization studies:

  • Use immunofluorescence microscopy with AtCPT7 antibodies coupled with markers for thylakoid sub-domains

  • Perform immunogold electron microscopy for high-resolution localization of AtCPT7 and its products

  • Employ biochemical fractionation to quantify distribution between stroma and membrane fractions

• Dynamic responses:

  • Monitor changes in AtCPT7 expression, localization, and activity under different environmental conditions

  • Analyze the temporal relationship between AtCPT7 activity and changes in thylakoid properties

This multifaceted approach will provide mechanistic insights into how AtCPT7-synthesized polyprenols contribute to thylakoid membrane structure and function.

What are the key controls needed when performing immunoblotting with At5g58770 antibodies?

Robust immunoblotting experiments with At5g58770 antibodies require multiple types of controls:

• Genetic controls:

  • Wild-type plants (positive control)

  • cpt7−/− knockout mutants (negative control)

  • RNAi lines with reduced AtCPT7 expression (for signal intensity correlation)

  • Overexpression lines for enhanced signal verification

  • Heterozygous plants from backcrosses for intermediate signal validation

• Technical controls:

  • Loading controls (housekeeping proteins) to normalize signal intensity

  • Secondary antibody-only controls to detect non-specific binding

  • Pre-immune serum controls when available

  • Blocking peptide controls to confirm epitope specificity

• Fraction purity controls:

  • When analyzing subcellular fractions, include markers for different compartments

  • For chloroplast work: stromal markers and thylakoid membrane markers

  • Assessment of fraction purity by immunoblot analysis using specific antibodies against known compartment markers

• Recombinant protein controls:

  • Purified recombinant AtCPT7 protein as positive control and standard

  • Truncated versions of AtCPT7 (e.g., AtCPT7Δ34N without the targeting sequence) to assess processing

The search results specifically mention the importance of assessing fraction purity through immunoblot analysis, highlighting this as a crucial control step .

How can At5g58770 antibodies be combined with other techniques to study polyprenol synthesis pathways?

Integrating At5g58770 antibodies with complementary techniques creates powerful research approaches:

• Combined antibody and activity assays:

  • Correlate AtCPT7 protein levels (detected by immunoblotting) with enzymatic activity

  • Measure product formation using techniques like thin-layer chromatography with 14C-labeled substrates

  • Connect protein expression with functional outcomes

• Multi-omics integration:

  • Combine immunodetection data with transcriptomics (RNA-seq) to assess post-transcriptional regulation

  • Integrate with metabolomics/lipidomics to correlate protein levels with polyprenol profiles

  • Link to phenotypic data to understand functional consequences

• Advanced microscopy approaches:

  • Co-localization studies using AtCPT7 antibodies with markers for different chloroplast compartments

  • FRET/FLIM techniques to study protein-protein interactions in the polyprenol synthesis pathway

  • Live-cell imaging to monitor dynamic changes in protein localization

• ChIP-based techniques:

  • ChIP-chip assays to study factors regulating AtCPT7 expression

  • CUT&RUN approaches to examine chromatin accessibility at the At5g58770 locus

  • Integration of epigenetic data with protein expression patterns

This integrative approach mirrors techniques described in search result , which details the use of multiple complementary methods including ChIP-chip, RNA-seq, CUT&RUN, and ATAC-seq for comprehensive analysis.

How can I resolve discrepancies between transcriptional and protein-level data for AtCPT7?

Researchers often encounter situations where mRNA levels do not directly correlate with protein abundance or activity. For AtCPT7 research, several methodological approaches can help resolve such discrepancies:

• Temporal analysis:

  • Perform time-course experiments to identify potential delays between transcription and translation

  • Sample at multiple timepoints after treatment or developmental changes

  • Compare the kinetics of mRNA change versus protein accumulation

• Post-transcriptional regulation assessment:

  • Analyze mRNA stability and half-life

  • Examine potential translational regulation mechanisms

  • Investigate protein turnover rates using cycloheximide chase experiments

• Compartment-specific analysis:

  • Separate analysis of different cellular compartments (whole cell vs. chloroplast fractions)

  • Account for protein redistribution between compartments

  • Consider protein processing effects (e.g., cleavage of transit peptides)

• Methodology validation:

  • Verify antibody detection limits and linearity range

  • Ensure RNA quantification methods are accurate and specific

  • Use multiple independent methods to confirm key findings

The research described in search result employed this multi-level approach, measuring AtCPT7 expression at the transcript level using quantitative RT-PCR while simultaneously assessing functional outcomes through polyprenol content analysis and phenotypic characterization.

What factors should be considered when troubleshooting weak or inconsistent AtCPT7 detection?

When experiencing weak or inconsistent detection of AtCPT7, consider the following factors:

• Sample preparation challenges:

  • Ensure complete protein extraction, especially for membrane-associated proteins

  • Optimize buffer conditions (detergents, salt concentration, pH)

  • Prevent protein degradation with appropriate protease inhibitors

  • Consider native versus denaturing conditions for membrane protein extraction

• Antibody-related factors:

  • Verify antibody storage conditions and activity

  • Optimize antibody concentration and incubation conditions

  • Test alternative antibodies targeting different epitopes

  • Consider potential cross-reactivity with other CPT family members

• Technical optimization:

  • Adjust protein loading to ensure detection within linear range

  • Optimize transfer conditions for membrane proteins

  • Test alternative detection methods (chemiluminescence vs. fluorescence)

  • Enhance signal using signal amplification techniques

• Biological considerations:

  • Account for developmental and tissue-specific expression patterns

  • Consider potential post-translational modifications affecting epitope recognition

  • Evaluate effects of environmental conditions on protein expression and stability

The search results note that AtCPT7 can form "discrete punctate structures" that are "characteristic of aggregated or misfolded proteins" under certain conditions , highlighting the importance of proper sample handling and preparation.

How should I interpret results from At5g58770 antibody studies in the context of protein truncation or modification?

The interpretation of immunodetection results for AtCPT7 requires careful consideration of potential protein processing events:

• Transit peptide processing:

  • AtCPT7 contains an N-terminal targeting sequence that directs it to chloroplasts

  • This transit peptide is typically cleaved upon import into chloroplasts

  • Antibodies targeting different regions might detect either the precursor, mature form, or both

  • The search results discuss several truncated versions including AtCPT7Δ34N (without targeting sequence) and further truncated versions

• Molecular weight considerations:

  • Compare observed molecular weights with predicted sizes for different forms

  • Account for post-translational modifications that might affect mobility

  • Consider potential degradation products or processing intermediates

• Domain-specific detection:

  • Antibodies targeting different domains may yield different results

  • N-terminal antibodies might not detect processed forms lacking the transit peptide

  • C-terminal antibodies may miss truncated products

• Experimental validation:

  • Use recombinant proteins with defined truncations as controls

  • Compare detection patterns across different genetic backgrounds

  • Perform mass spectrometry analysis to confirm protein identity and modifications

The search results specifically describe experiments with truncated versions of AtCPT7, including AtCPT7Δ34N and AtCPT7Δ7-67N, demonstrating that protein truncation can significantly affect function and localization .

How can bifunctional antibody approaches advance AtCPT7 research?

Bifunctional antibody technology, as referenced in search result , offers innovative approaches for studying AtCPT7:

• Custom specificity engineering:

  • Design antibodies with precisely controlled specificity profiles

  • Create reagents that can distinguish between closely related CPT family members

  • Develop antibodies that recognize specific conformational states of AtCPT7

• Advanced detection strategies:

  • Create bifunctional antibodies that simultaneously bind AtCPT7 and reporter molecules

  • Develop proximity-based detection systems for studying protein-protein interactions

  • Generate antibody-based biosensors that report on AtCPT7 activity or conformation changes

• Therapeutic and biotechnology applications:

  • Design antibodies that modulate AtCPT7 activity for studying functional consequences

  • Create tools for targeted protein degradation to achieve temporal control of AtCPT7 function

  • Develop antibody-based approaches for manipulating membrane properties in biotechnology applications

These approaches could be implemented using the biophysics-informed modeling framework described in search result , which combines "extensive selection experiments" with computational modeling to design antibodies with customized specificity profiles.

What epigenetic approaches can enhance our understanding of At5g58770 regulation?

Epigenetic regulation is a critical aspect of gene expression control. For studying At5g58770, several approaches can provide valuable insights:

• Chromatin modification analysis:

  • Examine histone modifications associated with At5g58770 expression

  • Use techniques like CUT&RUN to analyze specific modifications (H3K4me3, H3K9ac, H3K27me3)

  • Correlate modification patterns with expression levels across conditions

• Chromatin accessibility profiling:

  • Implement ATAC-seq to identify accessible regions in the At5g58770 locus

  • Analyze chromatin accessibility dynamics during development or stress responses

  • Integrate accessibility data with transcription factor binding information

• Transcription factor analysis:

  • Identify transcription factors that regulate At5g58770 expression

  • Use ChIP-chip or similar techniques to map binding sites in the promoter region

  • Analyze motif enrichment to identify key regulatory elements

• Data integration approaches:

  • Combine epigenetic data with transcriptomic and proteomic measurements

  • Develop comprehensive models of At5g58770 regulation

  • Identify key regulatory nodes that control expression under different conditions

Search result describes multiple epigenetic techniques including CUT&RUN and ATAC-seq that have been successfully applied to study gene regulation, providing a methodological framework for similar studies of At5g58770.