Biomedical Engineering Reference
In-Depth Information
To dishes washed with EBSS, add:
Starved: add 1 ml EBSS.
Starved plus BafA: EBSS plus 100 nM bafi lomycin A1.
Return to incubator and incubate for 90-120 min.
See
Note 4
for alternative treatments to induce or inhibit
autophagy.
4. Wash cells 3 times with PBS at 4°C. Remove all of the fi nal
PBS wash, and lyse each well in 100
L ice-cold lysis buffer
with dishes containing cells placed on ice.
5. Clear lysate by spinning for 5 min at 5,000 ×
g
. Remove and
keep 80
ʼ
L of the supernatant, toss pellet.
6. Add 5× sample buffer and incubate at 65 °C for 10 min
(
see
Note 9
).
7. Load the maximum sample volume (
see
Note 10
) (Invitrogen
Bis-Tris 4-12 % 20 well gels have a maximum volume of 25
ʼ
L,
thus the need to keep lysis volume to a minimum). Running
buffer for Bis-Tris gels is MES buffer.
8. Semi-dry transfer overnight, especially if you use a 1.5 mm gel
(
see
Note 11
).
9. Stain membrane with Ponceau S or similar protein stain to
check loading.
10. Block membrane with blocking buffer for 1 h at room tem-
perature. The membrane can be cut horizontally at different
parts to allow Western blotting of proteins with different
molecular weights with different antibodies. If only looking at
LC3 and actin/tubulin in the experiment, use the 30 kDa
molecular weight marker to separate membrane into two
halves. Top half is for actin/tubulin and bottom half for LC3.
11. For the optimum LC3 staining and quantifi cation using
LI-COR infrared fl uorescence dye, use anti-LC3 5F10 at
0.5
ʼ
g/ml in PBS, overnight at 4 °C. For ECL use the 5F10
at 0.5
ʼ
ʼ
g/ml or the polyclonal LC3 at 0.4
ʼ
g/ml. Also, probe
for actin or tubulin as a loading control.
12. For LI-COR infrared fl uorescence dye analyses, wash mem-
brane 3 times, 10 min each with PBST at room temperature.
For enhanced chemiluminescence (ECL) analysis, wash 3
times, 10 min each with PBST.
13. To detect LC3, incubate with LI-COR secondary antibodies
or HRP-conjugated secondary antibodies previously validated
in the laboratory. For actin or tubulin, use an antibody previ-
ously validated in the lab. Incubate for 1 h at room tempera-
ture or overnight at 4 °C.
14. Wash membrane 3 times, 10 min each with PBST, and then 2
times, 5 min each with PBST.
