lasting approximately 7 days. It is mandatory to daily check the
food intake and body weight.
- The specificity of dissection can be tested by detection of
contamination from adjacent nuclei by measurement of specific
markers of each nucleus, such as steroidogenic factor-1 (SF1) as
a marker of the VMH, proopiomelanocortin (POMC) for the
ARC, or orexin for the LHA. Analysis should be carried by
RT-PCR [40]
- For the whole hypothalamus (rat/mouse), use 400/200μL
BLYSpi, while for a nucleus-specific dissection, use
200/100μLBLYSpi.
- Increase time if necessary. It is very important to complete the
disaggregation of the tissue in order to achieve a high protein
concentration. With low volumes of BLYSpi, some foam may
form. Centrifuge the tubes for 10 s at maximum speed in a
benchtop centrifuge. You should be able to see the clear
homogenate with no bubbles. We recommend using safe-lock
tubes.
- It is recommended putting all tubes in the same orientation
into the centrifuge. This way it is possible to know the position
of the pellet in the tube even if it is not possible to see it.
- If the protein concentration is too low, it is possible to increase
the final volume of the loading, i.e., without altering the
amount of protein loaded to the gel.
- During the electrophoresis, the migration front can fade. This
may be due to a change in the pH of buffers or to abrupt
temperature changes.
- Do not let the filter paper or the membranes become dry. Wet
with more transfer buffer 1when necessary.
- Avoid using milk-based blocking since it may contain
phosphatases.
- To detect changes in the phosphorylation of AMPKα at
Thr-172, we use a Cell Signaling Technology antibody (rabbit
monoclonal anti-phospho-AMPKα (Thr-172), Cat#2535S)
diluted 1:2000 for total hypothalamic (20μg of protein per
sample) detection or 1:1000 (10μg of protein per sample) for
nucleus-specific detection. pAMPK is normalized withβ-actin
protein levels, although AMPKα1 and AMPKα2 (or total-
AMPK) levels should always be displayed. To detect the levels
of AMPKα1 and AMPKα2, we use Merck Millipore antibodies
(rabbit polyclonal anti-AMPKα1 and anti-AMPKα2, Refs
07-350 and 07-363, respectively) diluted 1:1000. To detect
the total levels of AMPKα, we use a Cell Signaling Technology
antibody (rabbit polyclonal anti-AMPKα, Cat#2532) diluted
1:1000.
Methods for Brain AMPK 445
