AMPK Methods and Protocols

5. Semidry blotting apparatus.


6. Tank blotting system. Store cooling element at 20 C.


7. Tris-buffered saline (TBS): 50 mM Tris–HCl, pH 7.5,
   150 mM NaCl.


8. TBS-T: TBS, 0.05% (v/v) Tween-20.


9. IB blocking solution: TBS-T, 5% (w/v) skim milk powder.
   Store at 4C.


10. IB antibody diluent solution: TBS-T, or TBS-T, 5% (w/v)
    BSA, or TBS-T, 5% (w/v) skim milk powder. Store at 4C.


11. Imager for recording of ECL signals.


12. ECL substrate solution.


13. ImageJ software (National Institute of Health, Bethesda).

2.5 Antibodies
and Conjugates

Antibodies listed below have been successfully used in this method

(seeNote 2).

1. Anti-ACC (Cell Signaling, 3676): for IB 1:1000 in TBS-T, 5%
   (w/v) BSA (seeNote 3).


2. Anti-ACC pSer79 (Cell Signaling, 11818): for IB 1:1000 in
   TBS-T, 5% (w/v) BSA (seeNote 4).


3. Anti-actin (C4, MP Biomedicals, 0869100): for IB 1:200,000
   in TBS-T.


4. Anti-AMPKα(Cell Signaling, 5831): for IB 1:1000 in TBS-T,
   5% (w/v) BSA (seeNote 3).


5. Anti-AMPKαpThr172 (Cell Signaling, 2535): for IB 1:1000
   in TBS-T, 5% (w/v) BSA (seeNote 4).


6. Anti-LC3 (Cell Signaling, 2775): for IB 1:1000 in TBS-T, 5%
   (w/v) BSA.


7. Anti-LC3 (MBL, M152-3B): for IF 1:50 in PBS, 1% (v/v) goat
   serum.


8. Anti-p62 (Progen, GP62-C): for IB 1:1000 in TBS-T (see
   Note 5).


9. Anti-Raptor (Cell Signaling, 2280): for IB 1:1000 in TBS-T,
   5% (w/v) BSA (seeNote 3).


10. Anti-Raptor pSer792 (Cell Signaling, 2083): for IB 1:1000 in
    TBS-T, 5% (w/v) BSA.


11. Anti-ULK1 (Cell Signaling, 8054): for IB 1:1000 in TBS-T,
    5% (w/v) BSA (seeNotes 3and 6 ).


12. Anti-ULK1 pSer555 (Cell Signaling, 5869): for IB 1:1000 in
    TBS-T, 5% (w/v) BSA (seeNote 6).

Autophagy 377