curve dilution as described: 0.7 mg/mL¼ 350 μL of 1 mg/
mL standard in 150μL of distilled water; 0.5 mg/mL¼ 250 μL
of 1 mg/mL standard in 250μL of distilled water; 0.25 mg/
mL¼ 250 μL of 0.5 mg/mL standard in 250μL of distilled
water, 0.125 mg/mL¼ 250 μL of 0.25 mg/mL standard in
250 μL of distilled water, 0.0625 mg/mL ¼ 250 μLof
0.125 mg/mL standard in 250 μL of distilled water;
0.03125 mg/mL¼ 250 μL of 0.0625 mg/mL standard in
250 μL of distilled water. Make a dilution to quantify the
protein. Take 10μL of the supernatant and add 250μLof
distilled water. Vortex. Add 10μL of the standard curve (in as-
cendant concentration) and the diluted sample in duplicates in
a 96-well plate. Add 250μL of Bradford solution into each
standard and sample wells. Eliminate the bubbles. Incubate
protected from light 5 min at 37 C. Measure output
(OD 590 nm) on a microplate reader.3.3 6.5% Sodium
Dodecyl Sulfate-
Polyacrylamide Gel
Electrophoresis
- Mix 2.45 mL of H 2 O, 5.06 mL of buffer A, and 2.18 mL
acrylamide. Add 56.25 μL of APS 10% and 28.125μLof
TEMED.Vortexitandcastgelwithina7.2cm10cm1.5mm
gel cassette. Fill to 0.5–1 cm below bottom of the comb teeth
and fill up with 60% 2-propanol. Allow polymerization and
remove 60% 2-propanol. Rinse the gel with distilled water to
wash residual 2-propanol and let it dry. - Prepare the stacking gel by mixing 1.77 mL of distilled water,
2.5 mL of buffer B, and 0.65 mL of acrylamide. Add 25μLof
APS 10% and 12.5μL of TEMED. Vortex it. Insert a 15-well
comb immediately without introducing air bubbles. - Prepare aliquots. Calculate the amount of protein of the sam-
ples extrapolating the ABS with the standard curve, and pre-
pare aliquots for WB mixing sample, loading buffer 1and
BLYSpi. For the hypothalamus, calculate volume of protein
homogenate to reach 20μg of protein (10μg if analyzing a
hypothalamic-specific nuclei), loading buffer 3.2μL (loading
buffer is prepared at 5but is use at 1; thus, the volume of
loading buffer is calculated by dividing the final volume by 5),
complete the volume up to 16μL with BLYSpi, and vortex (see
Note 10). - Heat the aliquots at 95C for 5 min. Centrifuge the heated
samples at 13,200 rpm for 9 s to bring down the condensate.
Perform the electrophoresis in ice or in a cold chamber at 4
C. Fill the mini-PROTEAN electrophoresis cell with 1 L of cold
running buffer. Pull comb straight up to remove. Load 5μLof
protein ladder (pre-thaw; do no heat). Add 16μL of protein
aliquots in the following wells. Run the electrophoresis at a
constant voltage of 140 V and 108 mA, and let the migration
front reach the 0.5 cm to edge of the gel (seeNote 11).
442 Patricia Seoane-Collazo and Miguel Lo ́pez