3.4 Semi-dry
Transfer
- Prepare the filter paper (extra-thick blot paper) and submerge it
into transfer buffer 1. Activate the PVDF membranes by
submerging them first in methanol, secondly in distilled
water, and thirdly in transfer buffer 1(5 min in each). - Put the filter paper in the Trans-Blot semi-dry transfer and then
the PVDF membranes, the gels, and on top of it another filter
paper. Remove the air bubbles in each step (seeNote 12). - Perform the transfer in constant amperage of 0.25 A and 25 V
approximately during 1 h and 40 min. - Block the membranes with BSA 3%-TBST 1, in agitation at
RT (seeNote 13).
3.5 Immuno-
detection
- Remove the blocking and incubate the membranes during 3 h
(or overnight) in agitation in the cold chamber (4C) with
primary antibody (seeNote 14). - Remove the primary antibody.
- Perform three washes in agitation in TBST (5 min each).
- Incubate the membranes during 1 h with secondary antibody
in agitation at RT. - Remove the secondary antibody.
- Perform three washes in agitation in TBST (5 min each).
- Prepare the ECL with a dilution of 1:1. Incubate the mem-
brane with ECL during 4 min protected from light. Remove
the excess ECL and put the membrane in a cassette between
acetate papers. Remove the air bubbles. - Develop in a dark chamber.
3.6 Analysis 1. Scan the autoradiographic images with a resolution of 800 dpi,
and save in TIFF format (seeFig. 2).
- Quantify digital images withImageJ Software(National Insti-
tutes of Health, USA), which detects the number of pixels in all
the samples of the same autoradiographic plate. Draw a rectan-
gle, with the same dimensions in each case, enclosing the
signal, and over the adjacent background. Determine the opti-
cal density of the signal and subsequently correct it by the
optical density of its background value. Express the data in
relation to a housekeeping protein (normally,β-actin for hypo-
thalamic samples) or the non-phosphorylated AMPKα (see
Note 15).
Methods for Brain AMPK 443