AMPK Methods and Protocols

20. Boil the samples for 5 min at 95C and place them on ice ready
    to be loaded onto an 8% polyacrylamide gel. We generally use a
    Mini-Protean electrophoresis system (BioRad), with a 10-well
    comb and a 1.5 mm glass plate, which allows the loading of the
    full sample (~60μl) per well. Run and transfer onto PVDF
    membrane as detailed elsewhere [33]. We generally perform
    the transfer for 75 min at 100 mV in transfer buffer.


21. Following 30 min of blocking with PBS-Tween-5% semi-
    skimmed milk, cut the membrane longitudinally in three pieces
    to separately blot overnight with antibodies against pAMPKα
    (Thr172, 1:1000, 62 kDa), pRaptor (Ser792, 1:1000,
    150 kDa), and pACC (Ser79, 1:500, ~280 kDa), in
    PBS-Tween-5% semi-skimmed milk.


22. The following day, wash the membranes three times with
    PBS-Tween for 5 min at RT and incubate with HRP-anti-
    rabbit IgG during 1 h. Following three further 5 min washes
    with PBS-Tween, develop with the ECL system following
    manufacturer’s instructions (seeNote 17).


23. Strip the antibodies from the membrane by incubating for
    10 min with re-blot solution.


24. Repeat previous steps using antibodies for analysis against
    AMPKα(1:1000), Raptor (1:1000), and ACC (1:1000).

3.4 Preparation
of Islet RNA for RNA-
seq (or qPCR)

We recommend the use of TRIzol reagent (Invitrogen) for RNA

isolation from purified islets.

1. Handpick 20–100 islets (seeNote 9) into 1.5 ml tubes. Spin for
   2 min at 200 rcf at RT to remove any leftovers of medium.


2. Resuspend the islets in 250μl of TRIzol by pipetting up and
   down or briefly vortexing. Incubate for 10–20 min at RT. Sam-
   ples can be frozen at this point.


3. Follow manufacturer’s instructions for RNA extraction.
   Importantly, perform the optional step indicated in the manual
   of adding 30μg glycogen (or equivalent reagent, such as
   glycoblue) preceding the RNA precipitation. This step will
   increase the RNA yield, which is otherwise low from such a
   small number of islets.


4. When working with a small number of islets, it is common to
   obtain low RNA concentration of poor purity, as estimated by
   the ratio of absorbance A260/A280 by a NanoDrop Spectro-
   photometer. We have found that a simple way to improve
   sample purity is to reprecipitate the RNA (performed after
   DNase treatment if this step is required) as follows.


5. Top up the volume with H 2 O to 100μl.


6. Add 0.1 volumes of 3 mM sodium acetate (pH 5.2). Vortex.

424 Aida Martinez-Sanchez et al.