Biomedical Engineering Reference
In-Depth Information
15. Reveal using LI-COR or with ECL reagent and X-ray fi lm.
Ensure that exposures are in the linear range.
16. Quantify your bands using imageJ or MetaMorph, acquire,
and subtract background (BG) (
see
Note 12
). Measure LC3-I
(minus BG) (your top band running at about 16 kDa), actin
(minus BG), and LC3-II (minus BG) (running just under-
neath LC3-I).
Quantify LC3-II over actin (
see
Note 13
).
4
Notes
1. Mowiol is prepared from a powder stock. Add 6 g of 100 %
glycerol to a 50 ml conical tube. Add 2.4 g Mowiol 4-88 and
mix well.
Add 6 ml of dH
2
O and mix. Allow to sit for 2 h at RT. Add
12 ml 0.2 M Tris-HCl, pH 8.5. Heat to 53 °C until Mowiol
has dissolved mixing occasionally. Aliquot and store at
−20 °C. Keep aliquots at 4 °C for immediate use.
2. Poly-
D
-lysine is essential for cells that are loosely attached
(especially during a 2 h amino acid starvation when they tend
to detach). Add 0.5 ml per well, covering the bottom of the
well or coverslip. Allow to sit for 5 min. Wash 3 times with
1 ml dH
2
O and once with 1 ml PBS.
3. Additional information about the maturation of the autopha-
gosomes can be obtained by using antibodies specifi c for
LAMP1, which is a lysosomal membrane protein found in
lysosomes and late endosomes. If the LC3-positive spots co-
localize with LAMP1, this indicates they have fused with late
endosomes and lysosomes and have become mature autopha-
gosomes or autolysosomes. We use a mouse anti-LAMP1 at
0.3
ʼ
g/ml that can be combined with rabbit anti-LC3 at
g/ml.
Note: optimization of concentration of primary antibody may
be required due to effi ciency of secondary antibodies.
4. Both rapamycin and Torin 1 can be added to normal growth
medium to induce autophagy, as they inhibit mTOR activity.
They can be combined with BafA to assess autophagy fl ux.
Wortmannin will inhibit production of PI3P and so also inhibit
formation of autophagosomes. All treatments should have a
similar effect of LC3 and WIPI2 spot formation. However,
LC3 remains associated with the autophagosomes until they
are consumed by lysosomes and thus marks all stages. WIPI2
dissociates from the autophagosomes before they become
LAMP1 positive.
0.4
ʼ
g/ml. The WIPI2 antibody should be used at 0.1
ʼ
