Customize GRF2 Antibody

Description

Introduction to GRF2 Antibody

GRF2 (guanine nucleotide-releasing factor 2) antibodies are essential tools for studying the molecular mechanisms of GRF2, a bifunctional guanine nucleotide exchange factor (GEF) that activates Ras and Rac GTPases. These antibodies enable the detection, quantification, and localization of GRF2 in various biological systems, including retinal development, cancer biology, and neurodegenerative disease research .

Key validation metrics for GRF2 antibodies :

Parameter	Details
Host Species	Rabbit polyclonal
Applications	Western Blot (0.04–0.4 µg/mL), IHC (1:50–1:200), IHC-Paraffin (1:50–1:200)
Specificity	No cross-reactivity with GRF1; validated via protein array (384 targets)
Storage	4°C short-term; -20°C long-term in 40% glycerol/PBS with 0.02% sodium azide

Retinal Photoreceptor Studies

GRF2 antibodies identified GRF2 as critical for:

Cone photoreceptor survival: GRF2-KO mice showed progressive cone degeneration .
Nuclear positioning: GRF2 regulates CDC42-dependent nuclear migration in the outer nuclear layer .
Synaptic integrity: Ribbon synapse flattening correlates with GRF2 loss .

Neurodegenerative Disease Models

Parkinson’s disease: GRF2 knockdown (via lentiviral shRNA) reduced ERK activation and FosB/ΔFosB expression in L-DOPA-induced dyskinesia .
Specificity: GRF1 and GRF2 have non-overlapping roles in striatal signaling pathways .

Cancer Biology

Triple-negative breast cancer (TNBC): Reciprocal GRF2/AnxA6 expression stratifies tumors into growth- vs. invasion-prone subtypes .
Therapeutic potential: High GRF2 correlates with rapid tumor growth, while low GRF2 predicts metastasis .

Technical Considerations

Ubiquitination assays: GRF2 degradation is Ras-dependent, with ubiquitination detectable via proteasome inhibition and anti-HA immunoblotting .
Co-immunoprecipitation: GRF2 antibodies confirmed interactions with Ras and Rac1 in retinal and neuronal tissues .
Limitations: Commercial GRF2 antibodies (e.g., Novus Biologicals™) require validation in species-specific models due to epitope variability .

Product Specs

Buffer

Preservative: 0.03% ProClin 300
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

Form

Liquid

Lead Time

14-16 weeks (made-to-order)

Synonyms

GRF2 antibody; GF14 antibody; At1g78300 antibody; F3F9.1614-3-3-like protein GF14 omega antibody; General regulatory factor 2 antibody

Target Names

GRF2

Uniprot No.

Q01525

Target Background

Function

This antibody targets a protein associated with a DNA-binding complex that interacts with the G-box, a well-characterized cis-acting DNA regulatory element in plant genes.

Gene References Into Functions

Relevant research indicates:

The phosphorylation status of Serine-62 regulates 14-3-3ω binding to target proteins. This suggests Serine-62 is a crucial element modulating target binding in both plants and animals. PMID: 26512969
The target protein is notable for possessing two independent 14-3-3 binding sites with differing affinities. Both sites require 14-3-3ω occupancy for complete inhibition of target protein activity under physiological conditions. PMID: 25578809

Database Links

KEGG: ath:AT1G78300

STRING: 3702.AT1G78300.1

UniGene: At.23337

Protein Families

14-3-3 family

Subcellular Location

Nucleus. Cytoplasm.

Q&A

FAQs for GRF2 Antibody in Academic Research

Advanced Research Questions

How does GRF2 regulate BCAR1-CRK-RAPGEF1 signaling in cell adhesion assays?
- Experimental design:
  - Use GRF2-specific siRNA or CRISPR-edited cells to assess changes in Rap1 activation via pull-down assays .
  - Combine immunofluorescence (IF) with GRF2 antibody to visualize colocalization with BCAR1/CRK complexes .
- Data contradiction analysis: Discrepancies in adhesion outcomes may arise from cell-type-specific post-translational modifications (e.g., phosphorylation at Ser/Thr residues) .

What controls are critical for interpreting GRF2 antibody specificity in flow cytometry?

Control matrix:

Control Type	Purpose	Example
Unstained	Baseline autofluorescence	Cells without antibody
Isotype	Nonspecific Fc binding	Rabbit IgG
Secondary-only	Secondary antibody noise	Anti-rabbit-AF488 alone
KO Validation	Target specificity	RAPGEF1-KO cells

How to resolve cross-reactivity of GRF2 antibodies with other guanine nucleotide exchange factors (GEFs)?
- Approach:
  - Perform sequence alignment of GRF2 (RAPGEF1) with homologous GEFs (e.g., GRF3/RAPGEF3) to identify divergent epitopes.
  - Validate using truncated mutants (e.g., aa 1–100 vs. 150–250) in dot blot assays .

Technical Optimization

What are the limitations of GRF2 antibodies in detecting phosphorylation-dependent interactions?
- Issue: Phosphorylation at Tyr/Ser residues (e.g., Tyr⁵⁰⁴) may alter epitope accessibility .
- Solutions:
  - Treat lysates with phosphatases (e.g., λ-PPase) to assess phosphorylation-dependent binding.
  - Use motif-specific antibodies (e.g., anti-pTyr) in parallel to correlate GRF2 activity states .
How to design functional assays for GRF2 in neurite outgrowth studies?
- Protocol:
  - Culture primary neurons with nerve growth factor (NGF) and inhibit GRF2 via siRNA.
  - Quantify neurite length and Rap1 activation using GRF2 antibody in WB/IHC .
- Data interpretation: Compare with RhoA pathway inhibitors to dissect GRF2-specific vs. RhoA-mediated effects .

Cross-disciplinary Considerations

Can GRF2 antibodies be used in structural studies (e.g., cryo-EM) for pathway mapping?
- Challenges: Low epitope accessibility in fixed, frozen samples.
- Workaround: Use nanobody-based labeling or epitope tagging (e.g., HA-tagged GRF2) for improved resolution .
How to reconcile conflicting reports on GRF2’s role in tumor metastasis?
- Analysis framework:
  - Context-dependent roles: GRF2 may act as a tumor suppressor in endothelial cells but promote invasion in epithelial cancers .
  - Meta-analysis of TCGA data stratified by RAPGEF1 expression and clinical outcomes.

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GRF2 Antibody