Customize lin-14 Antibody

Description

Definition and Mechanism

The lin-14 antibody specifically binds to epitopes in the LIN-14 protein, enabling its visualization or quantification in cellular contexts. LIN-14 is a nuclear protein with a BEN domain, functioning as a transcriptional regulator to control stage-specific gene expression during larval development . Its levels are tightly regulated by the lin-4 miRNA, which inhibits translation via complementary sequences in the lin-14 mRNA 3′ untranslated region (UTR) .

Key Features	Details
Target Protein	LIN-14 (heterochronic transcription factor)
Applications	Immunofluorescence, Western blotting, genetic rescue assays
Regulation	Downregulated by lin-4 miRNA; upregulated by lin-28 during early L1

Applications in Research

The lin-14 antibody has been pivotal in elucidating LIN-14’s role in developmental timing and gene regulation.

Immunofluorescence and Protein Localization

Used to track LIN-14 expression dynamics across larval stages. For example:

Wild-type: LIN-14 is abundant in L1 nuclei but declines by L2 .
lin-4 mutants: LIN-14 persists into L2, L3, and adult stages, causing reiteration of L1-specific lineages .
lin-28 mutants: LIN-14 levels drop prematurely, disrupting L1-specific cell fates .

Genetic Rescue Assays

LIN-14::GFP fusion constructs (validated via lin-14 antibodies) fully rescue lin-14 null mutants, confirming the protein’s nuclear localization and functional domains .

Western Blotting

Quantifies LIN-14 protein levels in mutants and wild-type animals, revealing:

lin-14(gf) mutants: Elevated LIN-14 persists beyond L1, altering developmental timing .
lin-4 mutants: LIN-14 accumulates due to miRNA-mediated translational inhibition .

Epitope Recognition

The antibody targets conserved regions in LIN-14’s C-terminal domain, which contains nuclear localization signals and DNA-binding motifs .

Control Experiments

lin-14(n536n540) null mutants: No staining observed, confirming antibody specificity .
LIN-14::GFP fusion: Co-localization with antibody signals validates epitope accessibility .

Temporal Regulation

Stage	LIN-14 Expression	Regulatory Mechanism
L1	High nuclear levels	lin-28 maintains expression
L2+	Undetectable	lin-4 miRNA inhibits translation

lin-4 mutants: LIN-14 persists post-L1, causing precocious cell lineage reiteration .
lin-28 mutants: Premature LIN-14 decline disrupts L1-specific differentiation .

Transcriptional Targets

LIN-14 directly regulates genes like ins-33 (insulin-like growth factor), as shown by DNA-binding assays and microarray data .

Cross-Species Conservation

LIN-14’s C-terminal domain is conserved in nematodes, suggesting functional homology in developmental timing .

Future Directions

ChIP-seq: Identify LIN-14 binding sites genome-wide to map direct targets.
CRISPR-Knockouts: Study LIN-14 interactions with miRNAs and other heterochronic genes.

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

lin-14 antibody; T25C12.1Protein lin-14 antibody; Abnormal cell lineage protein 14 antibody

Target Names

lin-14

Uniprot No.

Q21446

Target Background

Function

LIN-14 is a heterochronic protein that regulates the selection of stage-specific cell fates. It plays a role in the temporal progression of vulval fate patterning, potentially by inhibiting LIN-12. LIN-14 acts as a transcription factor, participating in the stage-specific repression of the insulin/insulin-like growth factor gene ins-33. It also contributes to the developmental timing of male tail tip morphogenesis and the regulation of seam cell development during larval stages. Furthermore, LIN-14 promotes survival at elevated temperatures in larval stages. It may specify L2 and later cell fates, acting as a temporal switch, and could potentially be involved in specifying L1 cell fates.

Gene References Into Functions

Research indicates that LIN-14 plays a crucial role in preventing axonal degeneration and maintaining the correct interaction between an axon and its surrounding tissue. PMID: 28930688
Findings suggest the importance of translational inhibition in silencing LIN-14 through the lin-4 microRNA. PMID: 24058689
By analyzing transcript counts in individual worms, it has been shown that pulsatile expression of the lin-4 microRNA maintains the temporal gradient of its target, LIN-14, by dampening its expression oscillations. PMID: 24036951
LIN-14 encodes a novel class of transcription factor. PMID: 16314527
Studies have revealed that lin-4 and lin-14 regulate lifespan in the adult organism. PMID: 16373574
Research demonstrates that nutrient deprivation leads to a loss of lin-14 mRNA, but not protein, repression. PMID: 19155321
Experiments have delineated a function for LIN-14 in coordinating the temporal progression of development, distinct from its role in regulating stage-specific events during the postembryonic development of C. elegans. PMID: 19161245

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Database Links

KEGG: cel:CELE_T25C12.1

STRING: 6239.T25C12.1a

UniGene: Cel.663

Subcellular Location

Nucleus. Note=May associate with mitotic chromosomes.

Tissue Specificity

High levels in hypodermal, intestinal, body wall muscle, nerve ring, and ventral nerve cord cells of embryos and L1 animals.

Q&A

FAQs for LIN-14 Antibody Research

Advanced Research Questions

What strategies resolve contradictions in LIN-14 localization studies?
LIN-14 exists in distinct nuclear populations: one DNA-associated and one free. To differentiate:
- Chromatin fractionation: Separate soluble and chromatin-bound fractions .
- Electrophoretic mobility shift assays (EMSA): Use recombinant LIN-14 C-terminal fragments to confirm DNA-binding activity (consensus sequence: 5'-CTGRT-3') .
How to design experiments identifying LIN-14 transcriptional targets?
- Microarray/RNA-seq: Compare mRNA profiles of wild-type, lin-14(lf), and lin-4(lf) mutants (elevated LIN-14) .
- Criteria for targets: Genes inversely regulated in lin-14(lf) and lin-4(lf) mutants (e.g., ins-33) .
- ChIP-seq: Use anti-LIN-14 antibodies to map DNA-binding sites .

What are common pitfalls in antibody-dependent LIN-14 studies?

Issue	Solution
Non-specific bands in Western blot	Optimize blocking buffers (e.g., 5% BSA + 0.1% Tween-20) .
Variable immunofluorescence signals	Standardize fixation protocols across replicates .
Batch-to-batch variability	Use RRID-tracked antibodies with publicly available validation data .

Methodological Best Practices

Antibody characterization: Follow multi-assay validation (Western blot, IP, IF) with protocols standardized across labs .
Data reporting: Include antibody lot numbers, dilution ratios, and negative controls in publications .
Consensus sequences: Refer to Kabat database alignments for cross-species antibody engineering insights .

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lin-14 Antibody