GID1C Antibody

What is GID1C and why is it important in plant research?

GID1C (Gibberellin Receptor GID1C) is one of three gibberellin (GA) receptors in Arabidopsis thaliana (the others being GID1a and GID1b). It functions as an ortholog of the rice GA receptor OsGID1, binding to GA molecules and showing particular affinity to GA4. This receptor is crucial in plant hormone signaling as it interacts with DELLA proteins in vivo in the presence of GA4, mediating gibberellin responses that control various aspects of plant growth and development . Understanding GID1C is essential for investigating hormone signaling mechanisms that regulate stem elongation, fertility, and other developmental processes in plants.

What are the alternative names and identifiers for GID1C protein?

GID1C is known by several alternative names in scientific literature, including AtCXE19, Carboxylesterase 19, GID1-like protein 3, and Protein GA INSENSITIVE DWARF 1C . For database referencing, researchers can use the UniProt identifier Q940G6 or the TAIR locus identifier At5g27320 . When searching literature or ordering antibodies, being aware of these alternative designations is important as different research groups and commercial providers may use varying nomenclature.

What are the basic properties of commercially available GID1C antibodies?

Commercial GID1C antibodies are typically polyclonal antibodies raised in rabbits using recombinant full-length Arabidopsis thaliana GID1c protein as the immunogen . These antibodies have a confirmed reactivity with Arabidopsis thaliana and predicted reactivity with related species like Arabidopsis alpina and Ricinus communis . They are primarily validated for Western Blotting applications with a recommended dilution of 1:5000, and the target protein has an expected molecular weight of approximately 38 kDa in Arabidopsis samples . Most commercial preparations are supplied in a lyophilized format requiring reconstitution with sterile water before use.

How should GID1C antibodies be stored and handled in the laboratory?

For optimal stability and performance, GID1C antibodies should be stored in lyophilized form at -20°C until needed . After reconstitution with the recommended volume of sterile water (typically 50 μL), researchers should prepare small aliquots to minimize freeze-thaw cycles, which can degrade antibody quality . Prior to opening the tubes, a brief centrifugation is recommended to collect any material that might adhere to the cap or sides of the tube, preventing sample loss . For long-term storage, reconstituted antibody should be kept at -20°C, while working aliquots can be maintained at 4°C for short periods during experimentation.

What is the optimal protocol for using GID1C antibody in Western blot applications?

For Western blot analysis using GID1C antibody, a standardized protocol begins with extracting approximately 20 μg of total protein from Arabidopsis thaliana tissues in 50 mM phosphate buffer (pH 7.0) containing protease inhibitors . The protein samples should be separated on an SDS-PAGE gel and transferred to a PVDF membrane. For optimal results, block the membrane with 2% ECL advance solution for 1 hour at room temperature with agitation . Incubate the blot with primary GID1C antibody at a dilution of 1:5000 (or 1:1000 for enhanced signal) overnight at room temperature with gentle agitation. After washing with TBS-T (one 15-minute wash followed by three 5-minute washes), incubate with HRP-conjugated secondary anti-rabbit antibody at a dilution of 1:75,000 for 1 hour at room temperature . Following the final wash series, develop the blot using a high-sensitivity chemiluminescence detection system.

Which plant tissues are most appropriate for GID1C antibody studies?

Based on experimental evidence, GID1C antibody studies can be effectively performed using protein extracts from various Arabidopsis thaliana tissues including cauline leaves, seedlings, and imbibed seeds . When designing comparative studies, it is important to consider the differential expression and function of GID1 receptors across tissue types. For example, when studying GA signaling in relation to stem elongation, seedling and mature stem tissues would be most appropriate since GID1a plays a primary role in this process . For fertility-related studies, reproductive tissues should be examined, where GID1b has demonstrated a more significant function in non-proteolytic GA signaling . Tissue selection should align with the specific GA-mediated developmental processes under investigation.

How can I validate the specificity of my GID1C antibody results?

To validate GID1C antibody specificity, multiple approaches should be implemented. First, include appropriate positive controls (wild-type Arabidopsis extracts) and negative controls (gid1c knockout mutant extracts if available) in Western blot analyses . Note that the antibody may recognize GST-GID1, which should be considered when working with recombinant proteins . Second, verify the observed molecular weight matches the expected 38 kDa size for Arabidopsis GID1C . Third, perform competition assays where pre-incubating the antibody with excess recombinant GID1C protein should diminish the signal. Fourth, validate results using alternative detection methods such as mass spectrometry or immunoprecipitation followed by Western blotting. Finally, compare your findings with published literature on GID1C expression and function to ensure biological relevance and consistency.

How can GID1C antibodies be utilized to investigate differential roles of GA receptors in proteolytic versus non-proteolytic signaling?

To investigate the distinct roles of GA receptors in different signaling pathways, researchers can design experiments comparing wild-type plants with various mutant combinations. Using GID1C antibody in conjunction with antibodies against GID1a and GID1b, Western blot analyses can be performed on protein extracts from single, double, and triple gid1 mutants in both wild-type and sly1 mutant backgrounds . In sly1 mutants, where DELLA proteins cannot be degraded, comparing GID1 protein levels with phenotypic outcomes helps distinguish non-proteolytic signaling contributions. Immunoprecipitation experiments using GID1C antibody can isolate GID1C-interacting proteins to identify unique binding partners in each signaling context. Additionally, chromatin immunoprecipitation sequencing (ChIP-seq) with GID1C antibody after crosslinking can reveal downstream transcriptional networks specific to proteolytic versus non-proteolytic pathways, providing deeper mechanistic insights into these distinct signaling modes.

What approaches can be used to study GID1C-DELLA protein interactions in planta?

For investigating GID1C-DELLA protein interactions in planta, several sophisticated approaches can be employed. Co-immunoprecipitation (Co-IP) using GID1C antibody can pull down native protein complexes from plant tissues treated with or without GA4, allowing detection of associated DELLA proteins by Western blotting . Bimolecular fluorescence complementation (BiFC) can visualize these interactions in living cells by fusing split fluorescent protein fragments to GID1C and DELLA proteins. Förster resonance energy transfer (FRET) microscopy offers quantitative assessment of protein proximity when GID1C and DELLA proteins are tagged with appropriate fluorophores. For temporal dynamics, researchers can combine GID1C antibody with proximity ligation assay (PLA) to detect transient interactions following GA treatment. These methods can be applied across various tissues and developmental stages in wild-type plants versus gid1 or sly1 mutants to elucidate context-specific interaction patterns related to different GA-mediated developmental processes.

How can quantitative analysis of GID1C protein levels inform our understanding of GA signaling dynamics?

Quantitative analysis of GID1C protein levels can provide critical insights into GA signaling dynamics across developmental stages and environmental conditions. Using GID1C antibody in quantitative Western blot analysis with appropriate loading controls and standard curves allows precise measurement of protein abundance changes . These measurements can be correlated with transcriptomic data to assess post-transcriptional regulation of GID1C. Time-course experiments following GA treatment can reveal rapid changes in receptor levels indicating feedback regulation. Comparing GID1C protein levels across wild-type, gid1a, gid1b, and sly1 mutant backgrounds helps elucidate compensatory mechanisms among GA receptors . Additionally, quantitative proteomics approaches combining immunoprecipitation with mass spectrometry can map post-translational modifications of GID1C that may regulate its activity or stability. This comprehensive quantitative analysis allows researchers to develop mathematical models predicting how receptor abundance influences signaling outputs under various physiological conditions.

What are common issues when working with GID1C antibody and how can they be resolved?

Several common issues may arise when working with GID1C antibody. For weak or absent signals in Western blots, first verify antibody viability by reducing dilution to 1:1000 and extending primary antibody incubation time . If background is excessive, increase washing duration and frequency, and optimize blocking conditions using different blockers like 5% BSA. When multiple bands appear, they may represent splice variants, degradation products, or cross-reactivity with other GID1 family members, which can be verified by including appropriate gid1 mutant controls . For inconsistent results between experiments, standardize protein extraction methods, ensure complete reconstitution of lyophilized antibody, and prepare fresh working solutions. If detecting GID1C in non-Arabidopsis species proves difficult despite predicted reactivity, consider using higher antibody concentrations and modified extraction buffers optimized for the specific plant species being studied . Always include positive controls from Arabidopsis samples when working with new species.

What statistical approaches are most appropriate for analyzing GID1C antibody-based Western blot data?

For robust statistical analysis of GID1C antibody-based Western blot data, researchers should employ multiple complementary approaches. Normalization to appropriate loading controls (like housekeeping proteins) is essential before any comparative analysis. For quantitative Western blots, densitometric measurements should be performed on multiple biological replicates (minimum n=3) to calculate means and standard deviations . Two-way ANOVA can assess the significance of treatment effects across different genotypes, while repeated measures ANOVA is appropriate for time-course experiments. For comparing multiple groups, post-hoc tests (such as Tukey's HSD) should follow ANOVA to identify specific significant differences. When analyzing ratios of GID1C to other proteins, log transformation before statistical testing often improves data normality. Power analysis should be conducted prior to experimentation to determine adequate sample sizes for detecting biologically meaningful differences. Finally, researchers should present both representative blot images and quantification graphs with clearly indicated statistical significance levels to enable proper interpretation by readers.

How do the functions of GID1a, GID1b, and GID1c differ in plant development and GA signaling?

The three GID1 receptors demonstrate distinct but overlapping roles in plant development and GA signaling pathways. In proteolytic GA signaling, GID1a serves as the primary receptor promoting stem elongation, while also playing a significant role in fertility . GID1c contributes secondarily to stem elongation in this context, with GID1b having minimal impact . In contrast, during non-proteolytic GA signaling (as observed in sly1 mutants), GID1b assumes a dominant role in fertility, with GID1a taking a secondary position . For stem elongation in non-proteolytic contexts, GID1a remains primary, but GID1b becomes more important than GID1c . This functional specialization may be partly explained by GID1b's higher affinity for DELLA proteins compared to GID1a and GID1c, making it particularly effective in non-proteolytic signaling where DELLA degradation doesn't occur . The differential expression patterns and binding affinities of these receptors likely evolved to provide plants with precise control over diverse GA-mediated developmental processes.

What is the recommended experimental design for comparing the three GID1 receptors in a single study?

A comprehensive experimental design for comparing the three GID1 receptors should incorporate multiple approaches. Genetically, researchers should analyze single, double, and triple gid1 mutants in both wild-type and sly1 mutant backgrounds to distinguish proteolytic and non-proteolytic signaling contributions . Phenotypic analyses should measure multiple GA-responsive traits including germination rates, stem elongation, and fertility across these genotypes. Biochemically, Western blotting with specific antibodies against each GID1 receptor should quantify protein levels across different tissues and developmental stages . In vitro binding assays can compare the affinity of each receptor for various GA molecules and DELLA proteins. Cell biology approaches should include subcellular localization studies using fluorescent fusion proteins to identify potential differences in receptor distribution. Finally, transcriptome analysis of the different mutant combinations can reveal receptor-specific gene regulation patterns. This multifaceted approach will provide a comprehensive understanding of the unique and overlapping functions of each GID1 receptor.

How can researchers effectively differentiate between GID1C and other GID1 receptors in their experiments?

To effectively differentiate between GID1C and other GID1 receptors, researchers should implement a multi-faceted strategy. First, verify antibody specificity through Western blot analysis of gid1 single, double, and triple mutants to confirm that each antibody recognizes only its target receptor . Second, design receptor-specific RT-qPCR primers at regions of low sequence homology to accurately quantify transcript levels for each GID1 gene. Third, when working with recombinant proteins, introduce epitope tags at divergent regions that won't interfere with function, enabling specific detection. Fourth, for imaging studies, create fluorescent protein fusions optimized to distinguish between the different receptors. Fifth, develop receptor-specific CRISPR-Cas9 strategies targeting unique regions of each gene for precise genetic manipulation. The table below summarizes key differentiating characteristics that can be exploited in experimental design:

By carefully considering these distinguishing features and implementing appropriate controls, researchers can confidently differentiate between the three GID1 receptors in their experimental systems.