For autophagy research

Anti-rat LC3, Rabbit

LC3 is a mammalian analogue of Atg8, which is a factor required for autophagy in budding yeast. It is synthesized in the cytoplasm and immediately undergoes C-terminal cleavage to form LC3-I. LC3-I then reacts with E1-like enzyme (Atg7) and E2-like enzyme (Atg3) and binds with phospholipids to form LC3-II. LC3-II then binds to autophagosome membranes. Therefore, LC3 is used as one of the markers of autophagy, and this product recognizes both LC3-I and LC3-II.

Product Information

  • Manufacturing Process: Synthetic peptides corresponding to the amino acid residues 5-18 of rat LC3B were coupled with KLH and raised in rabbits.
  • Product Form: Antiserum (does not contain preservatives or stabilizers).
  • Specificity: Reacts with human, rat, and murine LC3B
  • Working Dilution:
    Western blot 1:5,000
    Immunocytochemistry 1:200-500 (confocal microscopy)
  • Recommended Dilution Buffer:
    Western blot - 1 % BSA in 20 mmol/L Tris-HCl(pH 7.5), 0.15 mol/L NaCl, 0.1 % Tween20, 0.1 % NaN3
    Immunocytochemistry- 1 % BSA in 20 mmol/L Tris-HCl(pH 7.5), 0.15 mol/L NaCl, 0.1 % NaN3

Applications

Western blot of MEF extracts

00813_img01.jpg

Extracts of MEF were separated by SDS-PAGE and transferred to a PVDF membrane. Western blot was performed using this anti-rat LC3 as the primary antibody.


Primary Antibody: anti-rat LC3, Rabbit (1:5,000), room temperature, 1 hr
Secondary Antibody: HRP-conjugated anti-rabbit IgG, goat (1: 20,000), room temperature, 1 hr

(Data: by courtesy of Dr. Ueno, Department of Biochemistry (I), Graduate School of Medicine, Juntendo University)

Fluorescent Staining of HeLa cells

00813_img02.jpg

Sample: HeLa cells, fixed with paraformaldehyde and treated with digitonin
Blocking Solution: 20 mmol/L Tris-HCl(pH 7.5) containing 1% BSA and 1% normal sheep serum, 0.15 mol/L NaCl, 0.1% NaN3, 30°C, 1 hr
Primary Antibody: This anti-rat LC3, rabbit (1:500), 30°C, 1 hr
Secondary Antibody: Cy3-conjugated anti-rabbit IgG, sheep (1: 2,000), 30°C, 1 hr

(Data: by courtesy of Dr. Kikkawa, Biomedical Research Center, Graduate School of Medicine, Juntendo University)

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