FPS2 Antibody
Description
Definition and Biological Relevance of pS2
pS2 (TFF1) is a 6.5 kDa secreted protein belonging to the trefoil factor family, characterized by a disulfide-rich "trefoil domain" critical for epithelial protection and repair . It is estrogen-regulated in breast tissue and plays roles in mucosal healing, tumor progression, and response to endocrine therapies . pS2 expression is observed in 60% of breast carcinomas, particularly in estrogen receptor-positive tumors .
Key pS2 Antibodies: Characteristics and Applications
The following antibodies are widely used in research, with distinct epitope specificities and applications:
Positional Selectivity
Anti-pSer2 antibodies exhibit distinct affinities depending on the location of phosphorylated serine residues (pSer2) within the 52-repeat C-terminal domain (CTD) of RNA polymerase II:
-
E1Z3G: Highest affinity for pSer2 in central heptads (e.g., heptad 4, K<sub>d</sub> = 0.5 nM) vs. N-terminal heptad 1 (K<sub>d</sub> = 3.2 nM) .
-
EPR18855/2G1: Preferential binding to C-terminal heptads (e.g., peptide 37 showed 25–40× enhanced binding) .
Cross-Talk with Other Phosphorylations
Phosphorylation at adjacent residues modulates pSer2 recognition:
-
pSer7 (−2 position): Enhances E1Z3G and 2G1 binding by 4–6× .
-
pThr4/pSer5 (+2/+3 positions): Blocks recognition by EPR18855 and 2G1 .
Experimental Applications and Optimization
pS2 antibodies are validated for diverse techniques:
Critical Considerations for Experimental Design
-
Epitope Accessibility:
-
Cross-Reactivity:
-
Validation:
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
FPS2 Antibody catalyzes the sequential condensation of isopentenyl pyrophosphate with the allylic pyrophosphates, dimethylallyl pyrophosphate, and then with the resultant geranylpyrophosphate to the ultimate product farnesyl pyrophosphate.
- Farnesyl diphosphate synthase (FPS) single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal. PMID: 27382138
- Over-expression of FPS2 in the cytosol and targeting of its translated product to chloroplasts in Arabidopsis led to synthesis of several novel sesquiterpenes, including E-beta-farnesene, and induced alarm responses in Myzus persicae. PMID: 25538111
- Expression analysis of FPS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. PMID: 23145086
Q&A
Basic Research Questions
How to validate FPS2 antibody specificity in immunohistochemistry (IHC) and immunoblotting (IB)?
Validation requires a combination of controls:
-
Positive controls: Use tissues/cell lines with confirmed FPS2 expression (e.g., estrogen receptor-positive breast cancer samples) .
-
Negative controls:
| Control Type | Purpose | Priority | Source |
|---|---|---|---|
| Knockout tissue | Confirm absence of signal | High | |
| Peptide blocking | Verify target specificity | Medium | |
| Secondary-only | Detect background noise | High |
What experimental conditions optimize FPS2 antibody performance in flow cytometry?
-
Fixation: Avoid over-fixation with paraformaldehyde (>4% may mask epitopes).
-
Permeabilization: Use 0.1% Triton X-100 for intracellular staining.
-
Titration: Test antibody dilutions (1:50–1:500) using a cell line with known FPS2 expression (e.g., MCF-7) . Validate with fluorescence-minus-one (FMO) controls.
Advanced Research Questions
How to resolve conflicting FPS2 expression data between Western blot (WB) and IHC?
-
Post-translational modifications: FPS2 is glycosylated; use deglycosylation enzymes (e.g., PNGase F) in WB to confirm band identity .
-
Epitope accessibility: Paraffin embedding in IHC may obscure epitopes; antigen retrieval (pH 9.0 buffer) improves detection .
-
Quantitative comparison: Normalize IHC signal intensity to housekeeping proteins (e.g., β-actin) via multiplex fluorescence .
How to assess FPS2 antibody cross-reactivity in multi-omics studies?
-
Protein array screening: Test against >19,000 human proteins (HuProt™ array). Prioritize Z-scores >30 and S-scores >2.5 for specificity .
-
CRISPR validation: Knock out FPS2 in HEK-293 cells and compare signal loss to wild-type .
| Metric | Threshold | Interpretation |
|---|---|---|
| Z-score | >30 | Strong binding affinity |
| S-score | >2.5 | High target specificity |
How to integrate FPS2 antibody data with transcriptomic/proteomic datasets?
-
Orthogonal validation: Correlate IHC results with RNA-seq data for TFF1 (FPS2 gene) using platforms like TCGA.
-
Machine learning: Apply LASSO regression to identify confounding variables (e.g., sample fixation time) that affect antibody performance .
Troubleshooting Methodologies
How to address nonspecific bands in Western blotting with FPS2 antibody?
-
Pre-clearing: Incubate lysate with protein A/G beads before antibody exposure.
-
Alternative buffers: Replace TBST with high-stringency PBS-T + 0.5% casein.
-
Fragment analysis: Use reducing vs. non-reducing conditions to distinguish monomeric (7 kDa) vs. dimeric FPS2 .
What statistical approaches validate FPS2 as a biomarker in heterogeneous samples?
-
ROC analysis: Calculate sensitivity/specificity using clinical cohorts (e.g., breast cancer vs. normal tissue).
-
Meta-analysis: Aggregate data from public repositories (e.g., CPTAC) to assess consistency across platforms .
