Achemidoc imaging system

Embryos (10)
were incubated at 14 °C until 24 hpf and then collected
in a 1.5 mL microcentrifuge tube for each condition. Next, 200 μL
of HEPES buffer (20 mM, pH 7.3), supplemented with protease inhibitor
(Roche cOmplete, Mini Protease Inhibitor Cocktail), was added, and
embryos were lysed by pipetting up and down with a p200 tip about
15 times.^{90 (link)} The yolk platelets were pelleted
by centrifugation at 800 rcf for 10 min, and the supernatant was collected
into a new microcentrifuge tube. Lysates were centrifuged at 16 200
rcf for 5 min at 4 °C, and 30 μL of lysate was mixed with
10 μL of 4× SDS loading buffer, denatured at 95 °C
for 5 min, and loaded onto a 10% SDS polyacrylamide gel for SDS-PAGE
(150 V for 90 min). Protein was transferred to a PDVF membrane (80
V for 90 min), blocked with 5% BSA in TBST for 1 h at RT, and incubated
with either anti-HA rabbit monoclonal antibody (1:1000, CST #3724)
or anti-GAPDH rabbit polyclonal antibody (1:1000, Proteintech 50−172−604
6351) in 5% BSA TBST overnight at 4 °C. Blots were washed three
times with TBST and incubated with antirabbit monoclonal antibody-HRP
(1:1000 CST #7074) in TBST at RT for 1 h. Blots were washed three
times with TBST and then developed with SuperSignal West Pico Chemiluminescent
Substrate (Thermo) for 5 min before chemiluminescent imaging on a
ChemiDoc imaging system (Bio-Rad).