Autophagy pathway and target inhibitors

Combination of autophagy related Antibodies and Inhibitors

MBL provides a useful set of antibodies and inhibitors that can detect autophagy flux!

Anti-LC3 antibodies × 2 itemsLoading control antibodyPositive control for LC3Inhibitors × 2 itemsCell lysis buffer

Bafilomycin A1V type ATPase specific inhibitor

ChloroquineInhibitor of fusion between lysosome and autophagosome

It is possible to obtain these reagents in only one kit!

LC3

Atg5-Atg12 etc.Lysosome

Autophagosome

Autophagosome formation

Fusion with lysosome

Degradation of contents

AutolysosomeIsolationmembrane

Formation and elongation of isolation membrane

Hydrolase

Clone Isotype Applications ReactivityComponents Size

Anti-LC3 mAb-HRP-DirecTAnti-LC3 mAbAnti-α-Tubulin pAb-HRP-DirecTPositive control for anti-LC3 antibodyChloroquine solution(×1000)Bafilomycin A1 solution(×1000)Cell lysis buffer(×5)

8E104E12Polyclonal

Mouse IgG2aκMouse IgG1κRabbit IgG(aff.)

50 μl50 μl50 μl

100 μl100 μl100 μl

1 ml × 2

WBWB(weak), IC, IP, FCM, Immuno-EM

WBWB Positive Control

Hu, Mo, Rat, HmHu, Mo, Rat, HmHu, Mo, Rat, Hm,Chi

Kit components

Do you have any plans for watching autophagy in your research?

The kit includes HRP conjugated antibody for WB of autophagy flux assay. Realize time saving and brief operation!

Inhibitors are ready-to-use. Can be used by 1000-fold dilution!

Anti-LC3 antibody for immunocytochemistry is contained. Can visualize autophagosomes!

For More information go to the next page

Product informationCode Product Name

8486 Autophagy Watch

~for Autophagy Flux Assay~

For Research Use Only

AutophagyAntigen presentation

Cancer

Cell Death

Virus infection

Starvation adaptation

Bacterial infection

LC3 Atg16L

Beclinp62

Atg5 Neurodegerative disease

(Reference) N. Yoshimori, T. Mizushima. How to Interpret LC3 Immunoblotting. Autophagy 3, 542-545 (2007)

WB: Anti-LC3 mAb-HRP-DirecT

WB: Anti-α-Tubulin pAb-HRP-DirecT

20

15

75

50

37

1 2 3 4 5 6(kDa)

Nutrien

t3 h

starv

ation

3 h st

arvati

on +

Bafilom

ycin

A1

*Atg5

-/-MEF

Positiv

e con

trol

3 h st

arvati

on +

Chloroq

uine

LC3-ILC3-II

α-Tubulin

Cell: MEF

treatment(-) treatment(+)inhibitors(+)treatment(+)

LC3-II accumulates in the presence of inhibitors.

Inhibitor doesn’t affectthe amount of LC3-II.

The enhancement of the autophagic flux

LC3-I

LC3-II

* treatment ex.) starvation, knockout, etc.

How to Interpret LC3 Western blotting

Example of WB data by Autophagy Watch

Example of IC data by Autophagy Watch

Starvation induces increasing the amount of LC3-II. (lane1 vs. lane 2)Additionally, the amount of LC3-II increases in the presence of Chloroquine or Bafilomycin A1.(lane 2 vs. lane3, 4)These results suggest the accumulation of LC3-II under starvation condition indicates the enhancement of the autophagic flux.

* Atg5-/- MEF was kindly provided by Dr. Mizushima Noboru.(Department of Biochemistry and Molecular Biology Graduate School and Faculty of Medicine, The University of Tokyo)

Nutrient 3 h starvation 3 h starvation + Chloroquine 3 h starvation + Bafilomycin A1

Autophagy Watch i n c l udes Anti-LC3 mAb (clone: 4E12) for immunocytochemistry.This ant ibody can detect the accumulation of autophagosomes.

Microscope: BZ-9000 Generation II(Keyence), Cell:MEF

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