protocol evaluates the final global incorporation of labeled
phenylalanine into proteins. It integrates both entry and
removal of labeled phenylalanine all along the incubation
period. The longer your incubation time, the more your evalu-
ation will take into account putative protein degradation
(including autophagy).- Use adequate trash for liquid and tips due to the presence of
radioactivity. - Pellets do not have to be completely dissolved.
- Formic acid must be added under a hood because of its toxicity.
- Formic acid allows dissolution of the pellet.
- Such Western blot analysis can be also performed on cardio-
myocytes treated with radiolabeledL-phenylalanine (divided
after lysis into two samples, one for protein synthesis analysis
and the other for Western blot analysis). However, in this case,
all materials should be dedicated for radioactive samples. - This will prevent any putative dephosphorylation of the pro-
teins evaluated for its phosphorylation level. - It is better to perform two different blots in order to detect
phosphorylation and total proteins than to do stripping of a
same blot. Indeed, blot stripping is rarely 100% efficient and
frequently unequal (principally for the primary antibody) and
can falsify data obtained after reprobing with a second antibody
detecting the same protein on another epitope (total
vs. phosphorylation or vice versa) with the possibility of closed
epitope and evident steric hindrance. However, each blot is
tripped and reprobed with an antibody directed against a
housekeeping protein for which the protein level is known to
be unmodified by the different treatments and with a molecular
weight sufficiently different form the original target to be easily
differentiated (in the present cases, we used GAPDH or eEF2).
The data obtained with this loading control will be used to
normalize original data. - The black side of the holder cassette has to face the black side of
the container, and the white side has to face the red one. - For a more complete evaluation, you can also follow the phos-
phorylation state of both p70S6K and eEF2 in a time course
experiment all along the 24 h incubation time with hypertro-
phic agents and AMPK activators. - PFA in excess can be removed with a P1000 pipette.
- Cells have to be on the bottom side facing the antibody
solution. - Cells have to be on the top side to allow their correct washing.
340 Florence Mailleux et al.