leo-1 Antibody
Description
Introduction to LEO1 and Its Antibodies
LEO1 (RNA polymerase-associated protein LEO1) is a core component of the PAF1 complex (PAF1C), critical for transcription elongation, chromatin modification, and DNA repair . LEO1 antibodies are polyclonal rabbit-derived tools used to study this protein’s functions across species (human, mouse, rat) . These antibodies enable detection and analysis of LEO1 in research contexts, including Western Blot (WB), Immunohistochemistry (IHC), Immunoprecipitation (IP), and ELISA .
Key Features of LEO1 Antibodies
Western Blot (WB)
Immunohistochemistry (IHC)
-
Tissue Validation: Human colon, mouse renal cell carcinoma .
-
Antigen Retrieval: TE buffer (pH 9.0) or citrate buffer (pH 6.0) .
Immunoprecipitation (IP)
ELISA
Role in DNA Repair
-
CSB Interaction: LEO1 binds the C-terminal region of CSB, facilitating recruitment to transcription-blocking DNA damage sites (e.g., UV-induced cyclobutane pyrimidine dimers) .
-
Genotoxin Sensitivity: LEO1 deficiency increases sensitivity to UVC and cisplatin, impairing RNA synthesis recovery and CPD excision .
Transcription Elongation
-
PAF1C Complex: LEO1, alongside PAF1, regulates RNA Pol II degradation post-DNA damage via interactions with Ela1 .
-
Epigenetic Regulation: Mediates histone modifications (H2B ubiquitination, H3K4 methylation) and chromatin dynamics .
Cancer and Aging
-
Leukemogenesis: Collaborates with KMT2A/MLL1 rearrangements to promote oncogenesis .
-
Aging Pathways: Links epigenetic dysregulation to Cockayne syndrome-like phenotypes .
Optimal Dilution Ranges
Handling Notes
Product Specs
Composition: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
Q&A
How can researchers validate LEO1 antibody specificity across experimental models?
Validation requires parallel techniques to confirm target engagement. For WB, compare results across cell lines (e.g., HeLa, Jurkat) and tissues (mouse brain, human placenta) using knockdown controls . Proteintech’s polyclonal antibody (12281-1-AP) shows consistent reactivity at 105 kDa in WB across seven tissue types, but researchers should include siRNA-mediated LEO1 silencing to confirm band specificity . For IHC, antigen retrieval with TE buffer (pH 9.0) improves signal clarity in human colon tissues, while citrate buffer (pH 6.0) serves as an alternative .
Key Validation Data Table
| Application | Validated Models | Optimal Dilution | Observed MW |
|---|---|---|---|
| Western Blot | HeLa, HL-60, mouse brain | 1:500–1:2400 | 105 kDa |
| IHC | Human colon tissue | 1:50–1:500 | N/A |
| Immunofluorescence | HeLa cells | 1:10–1:100 | N/A |
What methodological considerations are critical for optimizing antibody dilutions?
Dilution ranges depend on target abundance and detection system sensitivity. For low-abundance nuclear targets like LEO1, higher concentrations (1:50 for IHC) are recommended . In Drosophila S2 cells, a 1:500 dilution of HA-tagged Leo1 antibody enabled ChIP-grade specificity without off-target binding . Titration curves should include a negative control (e.g., LEO1-knockout cells) to identify nonspecific signals.
How should researchers address discrepancies between observed and predicted molecular weights?
The 105 kDa observed MW (vs. 75 kDa predicted) arises from post-translational modifications (PTMs), such as phosphorylation or ubiquitination . To confirm, treat lysates with phosphatases (e.g., λ-PPase) and re-run WB. Alternatively, use mutagenesis to modify putative PTM sites in LEO1 and compare migration patterns.
What experimental strategies elucidate LEO1’s role in transcriptional regulation?
The PAF1C’s interaction with Myc provides a model for studying transcriptional recruitment:
-
Proximity-Ligation Assay (PLA): Detect Leo1-Myc interactions in fixed nuclei using HA-tagged Leo1 and endogenous Myc antibodies .
-
Re-ChIP Sequencing: Sequential immunoprecipitation of chromatin with anti-HA (Leo1) and anti-Myc antibodies identifies co-occupied promoters .
-
CRISPR-Cas9 Knockdown: Depleting Leo1 reduces Myc binding at promoter-associated E-boxes by 18%, quantified via ChIP-qPCR .
Transcriptional Impact of Leo1 Depletion
| Metric | Myc-Activated Genes | Myc-Repressed Genes |
|---|---|---|
| Median expression change (-Leo1) | 146% (vs. 176%) | 77% (vs. 57%) |
How can contradictions in subcellular localization data be resolved?
While LEO1 is primarily nuclear, cytoplasmic signals in IHC may arise from nonspecific binding or epitope exposure artifacts. To troubleshoot:
-
Use detergent-based permeabilization (0.5% Triton X-100) for nuclear-specific staining.
-
Validate with cytoplasmic-nuclear fractionation followed by WB .
What are the challenges in multiplex assays using LEO1 antibodies?
Cross-reactivity with secondary antibodies limits multiplex IHC/IF. Solutions include:
