Customize let-418 Antibody

Description

Introduction to LET-418 and Its Antibody

LET-418 (Lethal-418) is a Caenorhabditis elegans homolog of the Mi-2/CHD3 subunit within the NuRD complex, a chromatin remodeler critical for epigenetic regulation, DNA repair, and cell fate determination . The LET-418 antibody is a research tool used to study its protein-protein interactions, subcellular localization, and functional roles in developmental and cellular processes .

Role in Germ Cell Maintenance

LET-418 collaborates with the H3K4 demethylase SPR-5/LSD1 to maintain germline stem cell pluripotency. Mutations in let-418 and spr-5 lead to sterility, gonadal disorganization, and precocious reprogramming of germ cells into somatic fates, primarily neurons . This interaction counteracts COMPASS-mediated H3K4 trimethylation (H3K4me3), which correlates with somatic reprogramming .

Interaction with SPR-5/LSD1

Physical and genetic interactions between LET-418 and SPR-5 were confirmed via co-immunoprecipitation (co-IP):

LET-418 co-purifies with SPR-5 in wild-type extracts but not in spr-5-null mutants .
DNase/EtBr treatment does not disrupt the interaction, ruling out DNA-mediated binding .

Participation in NuRD and MEC Complexes

LET-418 defines two distinct chromatin-modifying complexes:

Complex	Components	Function
NuRD	HDA-1 (HDAC), LIN-53, LIN-40	Chromatin remodeling, deacetylation
MEC	MEP-1, HDA-1	Repression of germline gene expression in somatic cells

CHD-3, a paralog of LET-418, forms a separate NuRD complex and does not interact with LET-418 .

Meiotic Function

LET-418 and CHD-3 jointly ensure genome stability during meiosis by facilitating RAD-51 focus formation (a marker of homologous recombination). Their loss results in defective chromosome pairing and synapsis .

Co-Immunoprecipitation (Co-IP)

Purpose: Identify LET-418 interaction partners (e.g., SPR-5, MEP-1, HDA-1) .
Protocol:
1. Lysate preparation from wild-type or mutant C. elegans.
2. Antibody-based pull-down (e.g., anti-LET-418).
3. Western blot detection of co-purified proteins (e.g., SPR-5, HDA-1) .

Western Blot Analysis

Purpose: Validate protein expression or post-translational modifications.
Example: Anti-LET-418 antibodies detect LET-418 in extracts from mixed-stage worms .

Immunoprecipitation

Purpose: Study chromatin association or complex purification.
Key Findings: LET-418 interacts with HDA-1 in both NuRD and MEC complexes .

Table 1: LET-418 Interaction Partners

Partner	Complex	Function
SPR-5/LSD1	Independent	H3K4 demethylation, germ cell pluripotency
MEP-1	MEC	Repression of germline genes in somatic cells
HDA-1	NuRD/MEC	Histone deacetylation
LIN-53	NuRD	Chromatin remodeling

Table 2: Chromodomain Proteins and Histone Mark Correlations

Protein	Associated Histone Marks	Function
LET-418	H3K4me3, H3K9me2	Germ cell maintenance, meiosis
CHD-3	H3K4me3	Genome stability in meiosis
UAD-2	H3K27me3, H3K79me3	piRNA focus formation

Table 3: Experimental Validation via Western Blot

Target	Antibody	Observation
LET-418	Novus Biologicals	Detection in wild-type extracts, absent in mutants
HDA-1	Novus Biologicals	Co-purified with LET-418 in MEC/NuRD complexes
SPR-5	Anti-SPR-5	Co-IP with LET-418 in germ cell extracts

Product Specs

Buffer

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

Form

Liquid

Lead Time

Made-to-order (14-16 weeks)

Synonyms

let-418 antibody; evl-11 antibody; F26F12.7 antibody; Protein let-418 antibody; Lethal protein 418 antibody

Target Names

let-418

Uniprot No.

G5EBZ4

Target Background

Function

The LET-418 antibody targets a protein that is part of the NuRD (Nucleosome Remodeling and Deacetylase) complex. This complex is involved in the synMuv B pathway, which negatively regulates the specification of vulval cell fate in development. The negative regulation is believed to occur through interactions with the promoter of the lin-39 gene, a key regulator in vulva development. This process is dependent on the presence of the lin-1 gene. LET-418 also contributes to the negative regulation of the lag-2 gene, which is expressed in the gut during larval development. In addition to its role in vulval development, LET-418 has a broad role in development overall. In association with the akir-1 protein, LET-418 plays a role in regulating the transcription of antimicrobial peptide genes in response to fungal infections.

Gene References Into Functions

Suppressors of LET-418 complexes may represent direct competitors for chromatin regulation of critical genes involved in the transition to postembryonic development. PMID: 28007841
LET-418/Mi2 could be part of a system that drives development and reproduction with concomitant life-reducing effects later in life. PMID: 23815345

Database Links

KEGG: cel:CELE_F26F12.7

STRING: 6239.F26F12.7

UniGene: Cel.16862

Subcellular Location

Nucleus.

Tissue Specificity

Expressed in embryos and larva.

Q&A

FAQs for Researchers on LET-418 Antibody

Advanced Research Questions

How do LET-418-containing NuRD complexes differ from CHD-3-containing complexes?

Feature	LET-418 NuRD Complex	CHD-3 NuRD Complex
Core components	LET-418, HDA-1, LIN-53	CHD-3, HDA-1, LIN-53
Functional role	Germline integrity, somatic repression	Developmental patterning
Interaction partners	MEP-1 (MEC complex)	Not observed
Mutant phenotype	Sterility, somatic reprogramming	Viability, subtle morphogenesis defects

Methodological note: Use co-IP with anti-LET-418 antibodies to isolate complexes, followed by mass spectrometry for component identification .

How to resolve contradictions in LET-418’s role in H3K4 methylation regulation?
- Context-dependent effects: LET-418 suppresses H3K4me3 in the germline by counteracting COMPASS methyltransferase activity, but this varies by tissue .
- Experimental design:
  1. Perform ChIP-seq with anti-H3K4me3 antibodies in let-418 mutants vs. wild-type.
  2. Use tissue-specific reporters (e.g., nlp-29p::gfp) to assess spatial regulation .
  3. Combine with spr-5 mutants to dissect synergistic roles in epigenetic barrier maintenance .
What methods optimize LET-418 antibody use in co-IP under low-abundance conditions?
- Pre-clearing lysates: Reduce nonspecific binding using protein A/G beads prior to immunoprecipitation .
- Crosslinkers: Apply formaldehyde (1% for 10 min) to stabilize transient interactions .
- Validation: Include DNase/EtBr treatment to confirm interactions are not DNA-bridged (e.g., SPR-5/LET-418 interaction persists post-treatment) .

Data Contradiction Analysis

Why do some studies report LET-418 in both NuRD and MEC complexes?
- Complex plasticity: LET-418 dynamically associates with MEP-1 in the MEC complex for epidermal defense gene regulation, while HDA-1 anchors it to NuRD for germline functions .
- Resolution strategy:
  - Perform sequential IPs (e.g., anti-MEP-1 followed by anti-LET-418) to isolate subcomplexes.
  - Use tissue-specific promoters (e.g., dpy-7 for epidermis) to restrict analysis .

Methodological Best Practices

Antibody storage: Aliquot in 50% glycerol at -20°C to preserve activity.
Quantitative thresholds: For Western blots, use ≥10 µg of nuclear extract per lane to detect LET-418 reliably .
Ethical reporting: Disclose lot numbers and validation steps (e.g., mutant controls) in publications to enhance reproducibility.

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let-418 Antibody