- Prepare tissue lysis buffer (5 mL is sufficient for two whole
mouse kidneys). - Weigh empty sample tube, and then determine tissue weight.
- Calculate the required amount of lysis buffer for each tissue
sample; ideally there should be 0.5 g tissue per mL buffer. - Put tools on ice.
- Mince tissue in cold petri dish with razor blade until slurry is
produced. - Transfer slurry to mortar/pestle. Add predetermined tissue
lysis buffer volume and grind tissue carefully. Twist and pull
slowly until there are no visible tissue chunks remaining (clean
mortar/pestle with water between samples). - Transfer tissue to clean microfuge tube with a transfer pipet.
- Centrifuge at maximum speed (13,000 rpm or 16,000g) for
30 min at 4C(seeNote 20). - Transfer supernatant to a clean microfuge tube and discard
pellet. Keep supernatant on ice. - Prepare a 1:10 dilution of lysate (10μLin90μL tissue lysis
buffer) and measure protein concentration of dilution. Calcu-
late actual lysate concentration. - Aliquot samples and store at 80 C with protein concentra-
tion written on tube.
4 Notes
- When selecting rats, it is important to match the included rats
by weight and age if the different animal experiments are going
to be compared. Our studies have been carried out using male
Sprague-Dawley rats of ~250–300 g who are 8–12 weeks of
age. The key in selecting the animal model and animal char-
acteristics is that in order to compare results from a series of
replicate experiments, the animals used for one experiment
should be of the same sex and approximate age. - Two littermate mice of the same sex may need to be used for a
preparation if the number of conditions is more than four
to five. - Difficult to obtain in the US due to regulatory hurdles.
- Pentobarbital sodium is difficult to procure, isoflurane may be
substituted the only caveat is that the time under isofluorane
anesthesia must be kept constant for all the animals used for the
same study, as anesthetic gases may induce “preconditioning”
which makes the tissue resistant to ischemic insult, and is
equivalent to AMPK pre-activation [17]. Such preconditioning
460 Renee Rao et al.