PER2 Antibody

Basic Research Questions

How to validate PER2 antibody specificity for circadian rhythm studies?

• Method: Use knockout (KO) models as negative controls. In glial-specific Per2 KO mice, PER2 signals disappeared in GFAP⁺ astrocytes but remained in NeuN⁺ neurons, confirming antibody specificity for cell-type localization .

• Validation steps:

  Approach	Example from Studies
  Co-staining with cell markers	GFAP (astrocytes) and NeuN (neurons)
  Western blot MW verification	136 kDa observed vs. 137 kDa calculated
  KO tissue negative control	Absence of signal in conditional KO models

What experimental factors influence PER2 antibody performance in immunohistochemistry (IHC)?

• Critical variables:

  • Fixation time: Under-fixation causes signal diffusion; over-fixation masks epitopes. Studies used 4% PFA for 24 hr .

  • Antigen retrieval: Citrate buffer (pH 6.0) restored epitopes in FFPE sections .

  • Dilution optimization: Titrate between 1:200–1:500 for IHC .

Advanced Research Questions

How to resolve discrepancies in PER2 localization across studies?

• Case example: Nuclear vs. cytoplasmic PER2 localization varies by cell type and circadian phase .

  • Solution: Standardize Zeitgeber Time (ZT) for sampling. In alveolar type 2 (ATII) cells, PER2 peaks at ZT12 under intense light .

  • Methodological refinement:

    Step	Protocol
    Tissue collection	ZT3 for nuclear PER2 vs. ZT12 for cytoplasmic
    Co-staining markers	ProSP-C for ATII cells , GFAP for astrocytes

Can PER2 antibodies distinguish between isoforms or post-translational modifications?

• Limitations: Commercial antibodies (e.g., 20359-1-AP) target linear epitopes and may not differentiate phosphorylated isoforms .

• Alternate approaches:

  • Phospho-specific assays: Combine IP with Phos-tag® gels .

  • Circadian phase-specific sampling: Phosphorylation varies by ZT .

How to design PER2 functional studies in cell-type-specific contexts?

• Key findings:

  • ATII cells: Per2 deletion increased mortality during Pseudomonas aeruginosa infection (0% survival vs. 85% controls) .

  • Glial cells: Per2 KO altered mood-related behaviors without circadian disruption .

• Method: Use Cre-lox models (e.g., Sftpc-Cre for ATII cells) paired with PER2 antibody validation .

Methodological Challenges and Solutions

How to address cross-reactivity in non-model species?

• Data: Cited reactivity includes mouse, goat, and human , but validation required.

• Protocol:

  • Bioinformatic alignment: Compare immunogen sequence (e.g., human residues 751–850 ) with target species.

  • Negative controls: Use tissues from species-specific Per2 KO models.

What controls are essential for PER2 rhythm amplitude studies?

• Amplitude enhancement: Intense light increased PER2 trough/peak levels in ATII cells .

• Controls:

  • Light/dark cycles: 14h:10h vs. ambient light .

  • Blind mice: Eliminate light perception artifacts .

Data Interpretation Tables

Table 1: PER2 Antibody Performance Across Applications

Table 2: PER2 Functional Roles by Cell Type