GRHL1 Antibody

What is GRHL1 and what are its primary biological functions?

GRHL1 belongs to the grainyhead family of transcription factors that interact with sister of mammalian grainyhead (SOM) proteins. It functions as a transcription factor primarily involved in embryonic development, with two distinct transcript variants encoding different isoforms . Recent research demonstrates that GRHL1 plays significant roles in cell cycle regulation through transcriptional control of genes like CDC27, RAD21, CDC7, and ANAPC13, which are crucial for G2/M phase progression .

What detection methods can be used with anti-GRHL1 antibodies?

Anti-GRHL1 rabbit polyclonal antibodies can be utilized in multiple detection methods including:

• ELISA at a dilution of 1:62500

• Western blot at 1 μg/mL concentration (with HRP-conjugated secondary antibody at 1:50,000-100,000 dilution)

• Immunohistochemistry for tissue samples

The antibody is typically purified by peptide affinity chromatography and supplied in lyophilized form in PBS buffer with 2% sucrose at a final concentration of 1 mg/mL .

What is the recommended storage protocol for GRHL1 antibodies?

For optimal preservation of antibody activity, GRHL1 antibodies should be:

• Reconstituted with 50 μL of distilled water if supplied lyophilized

• Aliquoted to avoid repeated freeze-thaw cycles

• Stored at -20°C or below

• Handled with care to maintain the 1 mg/mL final concentration

How should researchers design experiments to investigate GRHL1's role in cell cycle regulation?

When investigating GRHL1's involvement in cell cycle regulation, researchers should implement a multi-faceted approach:

• Gene expression modulation:

  • Overexpression of GRHL1 using appropriate vectors

  • Knockdown using GRHL1-specific siRNAs

• Functional assays:

  • Cell proliferation assays to measure growth effects

  • Colony formation assays to assess clonogenic potential

  • Flow cytometry for cell cycle progression analysis (particularly focusing on G2/M phase arrest)

• Molecular analysis:

  • RNA sequencing to identify differentially expressed genes

  • qPCR verification of cell cycle-related target genes (CDC27, RAD21, CDC7, ANAPC13)

  • Western blot analysis of protein expression changes

What controls are essential when performing chromatin immunoprecipitation (ChIP) with GRHL1 antibodies?

When conducting ChIP experiments to identify GRHL1 binding sites:

• Input controls: Reserve a portion of pre-immunoprecipitated chromatin to normalize for differences in starting material

• Negative controls:

  • IgG control immunoprecipitation

  • Non-target genomic regions without predicted GRHL1 binding sites

• Positive controls: Known GRHL1 binding regions (e.g., promoters of CDC27, RAD21, CDC7, ANAPC13)

• Validation controls:

  • Luciferase reporter assays with wild-type and mutated binding sequences (e.g., mutating AACTAGTT to CTGTAGTT for CDC27)

  • GRHL1 knockdown/overexpression to confirm specificity

How does GRHL1 expression vary across different cancer types?

GRHL1 shows context-dependent expression patterns across different cancer types:

This heterogeneity suggests tissue-specific functions and regulatory mechanisms that require targeted investigation in each cancer context .

What is the relationship between GRHL1 and tumor immunology?

GRHL1 appears to play a significant role in tumor immunology, particularly in endometrial cancer where:

Researchers investigating tumor immunology should consider GRHL1 as a potential regulator of the immune microenvironment.

How can researchers investigate the upstream regulation of GRHL1?

To study the upstream regulation of GRHL1, researchers should consider:

• Signal transduction analysis:

  • Focus on the EGFR-ERK signaling axis, which has been shown to activate GRHL1

  • Investigate phosphorylation at Ser76, identified as a key regulatory site

• Experimental approaches:

  • EGF stimulation experiments to observe GRHL1 activation

  • Phosphorylation site mutants (e.g., S76A) to confirm functional relevance

  • Inhibitor studies targeting EGFR and ERK to confirm pathway specificity

  • Nuclear translocation assays to detect GRHL1 activation

• Technical considerations:

  • Use phospho-specific antibodies when available

  • Consider mass spectrometry to identify novel phosphorylation sites

  • Implement both in vitro kinase assays and cellular models

What approaches are recommended for analyzing GRHL1's impact on transcriptional networks?

For comprehensive analysis of GRHL1's transcriptional effects:

• Genome-wide binding profile:

  • ChIP-seq to identify global binding patterns

  • Motif analysis to refine understanding of binding preferences (known motifs include AACTAGTT, TACACGTT, AACATGTT, and GACACGTC)

• Transcriptional impact:

  • RNA-seq following GRHL1 modulation

  • GO and pathway enrichment analysis to identify biological processes affected

  • Integration with ChIP-seq data to distinguish direct vs. indirect targets

• Mechanistic validation:

  • Reporter assays with promoter constructs

  • Site-directed mutagenesis of binding sequences

  • Co-immunoprecipitation to identify protein interaction partners

What are common challenges when using GRHL1 antibodies in immunohistochemistry?

When performing immunohistochemistry with GRHL1 antibodies, researchers may encounter several challenges:

• Antigen retrieval optimization:

  • The search results indicate successful antigen retrieval using citrate buffer (pH 7.8, 0.1M) at approximately 82°C for 24 minutes

  • Different tissues may require adjusted protocols

• Staining optimization:

  • Consider overnight incubation with primary anti-GRHL1 antibody

  • Use biotin-conjugated secondary antibody (10-minute incubation at room temperature)

  • Implement streptavidin peroxidase treatment (5 minutes)

• Scoring considerations:

  • Quantity score (0-4): 0 (0%), 1 (1-10%), 2 (11-50%), 3 (51-80%), 4 (81-100%)

  • Intensity score (1-3): weak, moderate, strong staining

  • Calculate final IHC score by multiplying quantity and intensity scores

How can researchers address conflicting findings regarding GRHL1's role across different cancer types?

When encountering contradictory data about GRHL1's function:

• Context-specific analysis:

  • Explicitly define tissue and cancer type being studied

  • Consider histological subtypes that might have different GRHL1 functions

  • Account for tumor stage and grade in analyses

• Signaling context:

  • Evaluate status of upstream regulators (e.g., EGFR-ERK pathway)

  • Assess expression of known GRHL1 target genes

  • Consider potential compensatory mechanisms from other grainyhead family members

• Experimental validation:

  • Implement both gain-of-function and loss-of-function approaches

  • Validate findings in multiple cell lines representing the same cancer type

  • When possible, confirm in patient-derived models