GRF10 Antibody

What is GRF10 and why develop antibodies against this transcription factor?

GRF10 is a homeodomain transcription factor that separately regulates metabolic and morphogenesis genes in Candida albicans, a major human fungal pathogen. It controls purine and one-carbon metabolism in response to adenine limitation and is necessary for the yeast-to-hypha morphological switching, which is a known virulence factor . Developing antibodies against GRF10 enables researchers to:

• Study protein expression levels in different growth conditions

• Investigate protein-protein interactions, particularly with its binding partners

• Determine subcellular localization during various physiological states

• Analyze post-translational modifications that affect function

• Track morphological changes associated with virulence

GRF10 contains functional domains including activation domains in the C-terminal half and conserved interaction regions (IR) that make it an excellent target for antibody development to study transcriptional regulation mechanisms in pathogenic fungi .

What epitopes within GRF10 are most suitable for antibody development?

For effective antibody development against GRF10, researchers should consider targeting these specific regions:

When developing antibodies, researchers should avoid highly glycosylated regions and consider targeting residues D302 and E305, as mutations at these positions significantly affect transcriptional activity . Peptide-derived antibodies targeting these functional domains can provide valuable tools for studying GRF10's role in morphogenesis and metabolic regulation.

How can I validate the specificity of a newly developed GRF10 antibody?

Validating GRF10 antibody specificity requires a multi-step approach:

• Western blot analysis using genetic controls:

  • Wild-type C. albicans strain expressing native GRF10 (positive control)

  • GRF10 knockout strain (negative control)

  • GRF10 overexpression strain (increased signal intensity)

  • Testing with recombinant purified GRF10 protein

• Immunoprecipitation followed by mass spectrometry:

  • Confirm that the antibody pulls down GRF10 and its known interaction partners

  • Identify potential non-specific interactions

• Immunofluorescence microscopy:

  • Compare localization patterns in wild-type vs. GRF10 knockout strains

  • Use epitope-tagged GRF10 constructs as additional controls

• Cross-reactivity testing:

  • Test against closely related transcription factors with similar homeodomain structures

  • Examine reactivity across different Candida species and other fungi

Validation should include adenine-deprived and adenine-rich conditions to assess antibody performance across different GRF10 activation states .

How can GRF10 antibodies be used to study the dual functionality in metabolism and morphogenesis regulation?

GRF10 antibodies can be instrumental in elucidating the dual functionality of this transcription factor through several methodological approaches:

• Chromatin Immunoprecipitation (ChIP) studies:

  • Perform ChIP-seq experiments under both metabolic stress (adenine limitation) and morphogenesis-inducing conditions

  • Compare GRF10 binding patterns to identify distinct sets of target genes

  • Protocol should include formaldehyde crosslinking (1% for 15 minutes), sonication to 200-500bp fragments, and immunoprecipitation with anti-GRF10 antibodies

• Co-immunoprecipitation paired with proteomic analysis:

  • Identify different protein interaction partners under various conditions

  • Compare GRF10 interactomes during metabolic regulation versus morphogenesis

  • Example workflow: lyse cells in non-denaturing buffer, pre-clear with protein A/G beads, incubate with anti-GRF10 antibody, wash stringently, and analyze by LC-MS/MS

• Proximity-dependent biotin labeling:

  • Create a GRF10-BioID or GRF10-APEX2 fusion protein

  • Use anti-GRF10 antibodies to validate expression and functionality before biotin labeling experiments

  • Identify condition-specific proximal proteins

The experimental evidence from the search results indicates that GRF10 separately regulates metabolic and morphogenesis genes, with domain-specific functionality. The LexA-Grf10 fusion protein activates the lexA op-HIS1 reporter in an adenine-dependent fashion, yet its filamentation-inducing activity when overexpressed requires a functioning homeodomain but is independent of adenine levels .

What are the best approaches for using GRF10 antibodies to study post-translational modifications?

Post-translational modifications (PTMs) of GRF10 likely regulate its differential activity in metabolism versus morphogenesis pathways. The following approaches are recommended:

• Phosphorylation analysis:

  • Use phospho-specific GRF10 antibodies developed against predicted kinase target sites

  • Perform immunoprecipitation with general anti-GRF10 antibody followed by western blotting with phospho-specific antibodies

  • Compare PTM patterns under adenine limitation versus morphogenesis-inducing conditions

• Mass spectrometry-based PTM mapping:

  • Immunoprecipitate GRF10 using validated antibodies

  • Perform tryptic digestion and enrichment for modified peptides

  • Analyze by LC-MS/MS to identify and quantify PTMs

  • Expected workflow should include IMAC enrichment for phosphopeptides or antibody-based enrichment for other modifications

• In vitro kinase assays:

  • Immunoprecipitate GRF10 using specific antibodies

  • Incubate with candidate kinases and ATP

  • Analyze modification by western blotting or mass spectrometry

Based on the search results, particularly focusing on the temperature-responsive activation domain (AD1) and the critical residues D302 and E305, these sites should be primary targets for PTM analysis .

How can GRF10 antibodies be utilized to investigate protein-DNA interactions during morphogenesis?

Investigating GRF10's interaction with DNA during morphogenesis requires specialized applications of GRF10 antibodies:

• ChIP-seq during morphological transitions:

  • Induce filamentation through established methods (serum, temperature shift, N-acetylglucosamine)

  • Perform time-course ChIP-seq with anti-GRF10 antibodies at 15, 30, 60, and 120 minutes post-induction

  • Protocol modification: For fungi with cell walls, include zymolyase treatment (5 units/ml for 30 minutes) before lysis

  • Compare binding profiles to identify early vs. late target genes

• DNA affinity purification sequencing (DAP-seq):

  • Immunopurify GRF10 using anti-GRF10 antibodies

  • Incubate purified GRF10 with fragmented genomic DNA

  • Sequence bound DNA fragments to identify in vitro binding sites

  • Compare with in vivo ChIP-seq data to identify potential regulatory co-factors

• CUT&RUN or CUT&Tag approaches:

  • Use anti-GRF10 antibodies with protein A-micrococcal nuclease fusion

  • Perform in intact cells during morphological transitions

  • Advantage: Requires fewer cells than traditional ChIP

Research indicates that GRF10 overexpression leads to filamentation, and this requires a functioning homeodomain, consistent with GRF10 controlling the expression of key filamentation genes. This filamentation induced by GRF10 overexpression was independent of adenine levels and Bas1 , suggesting that the DNA-binding specificity of GRF10 changes during morphogenesis, which can be captured through these antibody-based approaches.

What are common challenges when using GRF10 antibodies for chromatin immunoprecipitation (ChIP)?

Researchers frequently encounter several challenges when using GRF10 antibodies for ChIP experiments:

• Low signal-to-noise ratio:

  • Root cause: The homeodomain structure of GRF10 shares homology with other transcription factors

  • Solution: Use highly specific monoclonal antibodies targeting unique GRF10 epitopes

  • Alternative approach: Employ a dual crosslinking protocol with DSG (2 mM for 45 minutes) followed by formaldehyde (1% for 15 minutes)

• Variable GRF10 expression levels:

  • Root cause: GRF10 expression changes with adenine availability and temperature conditions

  • Solution: Normalize ChIP data to input and GRF10 expression levels determined by western blot

  • Recommended control: Perform parallel ChIP experiments with an epitope-tagged GRF10 version

• Cell wall interference in Candida species:

  • Root cause: The fungal cell wall can impede antibody accessibility

  • Solution: Optimize spheroplasting conditions (Zymolyase 100T at 5 units/ml for 30 minutes at 30°C)

  • Critical step: Monitor spheroplasting microscopically to prevent over-digestion

• Crosslinking inefficiency:

  • Root cause: GRF10's interaction with DNA may be transient during certain phases

  • Solution: Test multiple crosslinking reagents and times

  • Optimal protocol: Based on the temperature-responsive nature of AD1 domain, use 1% formaldehyde for 15 minutes at experimental temperature rather than fixed temperature

The challenges stem from the context-dependent activity of GRF10, which regulates different sets of genes depending on metabolic conditions and morphogenesis signals, as indicated by the activation domains responding differently to temperature and adenine limitation .

How can I optimize immunofluorescence protocols for detecting GRF10 in different morphological forms of Candida albicans?

Optimizing immunofluorescence for GRF10 detection across different morphological forms requires modifications to standard protocols:

• Fixation optimization for different morphologies:

  • Yeast form: 4% paraformaldehyde for 30 minutes

  • Hyphal form: 4% paraformaldehyde + 0.1% glutaraldehyde for 45 minutes

  • Rationale: Hyphal forms require stronger fixation to preserve structure integrity

• Cell wall digestion adjustments:

  • Yeast form: Zymolyase 100T (2 units/ml) for 30 minutes

  • Hyphal form: Zymolyase 100T (5 units/ml) for 45 minutes + Chitinase (0.1 units/ml)

  • Critical control: Monitor cell wall digestion using calcofluor white staining

• Antibody penetration enhancement:

  • Increase detergent concentration in permeabilization buffer (0.5% Triton X-100)

  • Include 5% DMSO in antibody incubation buffer

  • Extend primary antibody incubation to overnight at 4°C

• Signal amplification strategies:

  • Employ tyramide signal amplification for low abundance detection

  • Use secondary antibodies conjugated to brighter fluorophores (Alexa Fluor 647)

  • Consider quantum dots for long-term imaging experiments

These optimizations address the challenges associated with GRF10's role in morphogenesis, as indicated by research showing that overexpression of GRF10 leads to filamentation and requires a functioning homeodomain .

How can I interpret conflicting data between GRF10 antibody-based assays and genetic expression studies?

When facing discrepancies between antibody-based detection of GRF10 and genetic expression data, consider these analytical approaches:

• Protein versus mRNA dynamics:

  • GRF10 protein levels may not directly correlate with mRNA levels due to post-transcriptional regulation

  • Action plan: Perform time-course studies measuring both mRNA (by RT-qPCR) and protein (by western blot) levels

  • Expected outcome: Possible time lag between transcriptional and translational changes

• Antibody epitope accessibility:

  • Underlying issue: Protein-protein interactions or PTMs may mask epitopes in certain conditions

  • Analytical approach: Use multiple antibodies targeting different GRF10 regions

  • Validation method: Compare native GRF10 detection with epitope-tagged versions

• Functional state versus abundance:

  • Conceptual framework: GRF10 activity may change without corresponding changes in protein levels

  • Experimental strategy: Complement antibody detection with reporter assays for GRF10 activity

  • Example: Compare ChIP-seq data with western blot quantification

• Context-dependent localization:

  • Mechanistic explanation: Nuclear versus cytoplasmic distribution may change in response to signals

  • Analytical method: Perform subcellular fractionation before western blotting

  • Critical control: Include markers for different cellular compartments

The search results support this approach by showing that GRF10 has context-specific functions - the LexA-Grf10 fusion protein activates transcription in an adenine-dependent fashion independent of Bas1, while its role in filamentation requires the homeodomain but is independent of adenine levels .

What advanced statistical approaches should be used when analyzing GRF10 ChIP-seq data?

Analysis of GRF10 ChIP-seq data requires sophisticated statistical approaches to account for its dual regulatory roles:

• Differential binding analysis:

  • Use DESeq2 or edgeR for comparing GRF10 binding profiles between conditions

  • Implement IDR (Irreproducible Discovery Rate) methodology for replicate consistency

  • Apply stringent FDR correction (q < 0.01) for peak calling across different conditions

• Integrative genomics approaches:

  • Correlate GRF10 binding with histone modification ChIP-seq data

  • Perform motif enrichment analysis within condition-specific peaks

  • Integrate RNA-seq data to correlate binding with expression changes

• Time-series analysis for morphogenesis studies:

  • Apply Hidden Markov Models to detect progressive binding changes

  • Use clustering approaches (maSigPro or ImpulseDE2) for temporal binding patterns

  • Perform gene ontology enrichment on time-specific gene clusters

• Multifactorial design considerations:

  • Account for both adenine availability and morphogenesis conditions

  • Implement ANOVA-like frameworks for multi-condition comparisons

  • Consider Bayesian approaches for complex experimental designs

This approach is supported by the finding that GRF10 separately regulates metabolic and morphogenesis genes, suggesting distinct binding patterns and regulatory mechanisms for these different functions .

How might GRF10 antibodies be used to study potential roles in host-pathogen interactions?

GRF10 antibodies can open new research directions for studying host-pathogen interactions:

• Monitoring GRF10 dynamics during host cell encounters:

  • Use immunofluorescence with anti-GRF10 antibodies during co-culture with host cells

  • Track subcellular localization changes during adherence and invasion

  • Correlate GRF10 activity with virulence-associated morphological transitions

• Extracellular GRF10 investigation:

  • Assess potential secretion/release of GRF10 during infection using sensitive immunoassays

  • Examine host immune recognition of GRF10 using antibody-based detection systems

  • Investigate whether host antibodies develop against GRF10 during infection

• Host environment sensing:

  • Study adenine-dependent regulation of GRF10 in different host microenvironments

  • Use ChIP-seq with GRF10 antibodies in infection models to identify in vivo targets

  • Examine temperature-responsive activation domains under host fever conditions

• Therapeutic targeting potential:

  • Develop neutralizing antibodies against critical GRF10 domains

  • Test inhibition of morphogenesis through GRF10-targeting approaches

  • Screen for small molecules that disrupt GRF10 function using antibody-based assays

This direction builds on the finding that GRF10 is necessary for the yeast-to-hypha morphological switching, which is a known virulence factor, and that its activation domains (particularly AD1) are temperature-responsive , suggesting potential adaptation mechanisms during host interaction.

What approaches could integrate GRF10 antibody techniques with CRISPR-based functional genomics?

Integrating GRF10 antibody techniques with CRISPR technologies offers powerful new research strategies:

• CUT&Tag-CRISPR screening:

  • Use GRF10 antibodies for CUT&Tag to identify binding sites

  • Target CRISPR interference or activation to these sites

  • Assess phenotypic outcomes to identify functionally important binding events

  • Protocol should include dCas9-KRAB or dCas9-VP64 constructs targeted to GRF10 binding sites

• Domain-specific perturbation analysis:

  • Generate precise mutations in functional domains (homeodomain, AD1, IR region)

  • Perform ChIP-seq with GRF10 antibodies on each mutant

  • Compare binding profiles to identify domain-specific regulatory networks

  • Validate findings by assessing morphogenesis and metabolic phenotypes

• GRF10 interactome perturbation:

  • Use CRISPR to knock out predicted GRF10 interaction partners

  • Perform immunoprecipitation with GRF10 antibodies followed by mass spectrometry

  • Identify changes in the GRF10 interactome and correlate with functional outcomes

  • Focus on interactions that respond to adenine limitation or temperature shifts

• Dynamic regulatory network mapping:

  • Create CRISPR-based fluorescent reporters for GRF10 target genes

  • Validate with GRF10 antibody ChIP-qPCR

  • Perform live-cell imaging during environmental transitions

  • Construct predictive models of GRF10-mediated gene regulation

This integration leverages the finding that GRF10 contains distinct functional domains, including temperature-responsive activation domains and conserved interaction regions with critical regulatory residues (D302 and E305) , which can be precisely targeted using CRISPR-based approaches and monitored using antibody-based techniques.