little as ten islets (< 100 μm) per lane when immunoblot was
performed using a WES system (http://www.proteinsimple.
com/wes.html) (unpublished data) as in [38]. Nevertheless,
detection of pRaptor with the antibody used for standard WB,
as described above, failed to provide a detectable signal
by WES.- AMPK activity is regulated by glucose, over the physiological
range of concentrations, in primary rodent and human islets of
Langerhans [24]. AMPK is strongly activated by glucose con-
centrations of 5.5 and 3 mM in mouse and human islets,
respectively (unpublished data and [24]). It is important to
adjust the glucose concentration at which the islets are main-
tained when performing experiments aimed to modulate
AMPK activity. This is due to the strong effect that glucose
has on the activity of this enzyme. We have found that AMPK
(as well as its targets ACC and Raptor) is strongly phosphory-
lated at concentrations<3mMor<5 mM in human and
mouse islets, respectively, while almost completely depho-
sphorylated at concentrations >11 mM or >17 mM in
human and mouse islets, respectively. Thus, we recommend
to perform experiments with AMPK-DN at low glucose con-
centrations and, conversely, at high glucose concentrations
with AMPK-CA. - Even though no major differences were observed inβ-cell mass
or the number of islets of AMPKdKO mice in vivo (unpub-
lished data and [7]), the yield of isolated islets was consistently
and substantially lower than that of the control (or of wild-type
animals of the same background-C57BL/6). We still don’t
know the cause of this difference, but we hypothesize that it
arises during the islet purification steps, possibly due to a
higher fragility of the islets. Whereas we are working toward
adapting the protocol to improve the islet yield from these
animals, this is currently of ~30–80 islets per mice. - If less than 10μg of total protein extracts are available, the assay
can be performed with as little as 2.5μg of protein, but reduc-
ing the amount of the other reagents by half and in a total
volume of 25μl. - If a very low number of islets are used, the pellet will be barely
visible. In that case, just add the loading dye to the whole
sample. If a high number of islets are used, the pellet might
be gelatine-like (high content of DNA), and the samples will
need to be handled with care to obtain a clean supernatant. In
that case, a longer centrifugation time may help or, alterna-
tively, it might be easier to pipette out the gelatinous pellet and
keep the rest of the sample (supernatant) in the original tube.
428 Aida Martinez-Sanchez et al.