The left eye was separated using protein extraction solution (iNtRON, Seoul, Korea), and the obtained protein was quantified using a BCA protein assay kit (BIO-RAD, Hercules, CA, USA). Equal amounts of protein were mixed 1:1 with sample buffer (Laemmli sample buffer (BIO-RAD) and 2-mercaptoethanol (BIO-RAD) mixed at 19:1) in each group and then heated at 99°C for 5 minutes. Heated protein was electrophoresed via 12% SDS polyacrylamide gel and transferred by electrophoretic means to nitrocellulose membrane. The membrane was blocked with 5% skim milk at room temperature, and then primary antibodies against the following proteins were used: rhodopsin (Abcam, Burlingame, CA, USA), opsin (Millipore, Burlington, MA, USA), heme oxygenase (HO-1, Cell Signaling, Beverly, MA, USA), nuclear enzyme erythroid 2-related factor-2 (NRF2, Santa Cruz Biotechnology, Santa Cruz, CA, USA), caspase 3 (Cell Signaling), and poly (ADP-ribose) polymerase (PARP, Cell Signaling). Horseradish-peroxidase-conjugated anti-mouse, and anti-rabbit secondary antibody were diluted in TBS-T solution. All western blots were re-probed with anti-β-actin antibody (Cell Signaling) to ensure protein loading. Bands were visualized with EZ-capture ST (ATTD) reagents according to the manufacturer's instructions.
Protein extraction solution
Manufactured by Bio-Rad
Protein extraction solution is a reagent designed to isolate and extract proteins from biological samples. It functions by disrupting cellular membranes and releasing proteins into the solution, enabling their subsequent analysis or purification.
Automatically generated - may contain errors
Lab products found in correlation
2 mercaptoethanol, by Bio-Rad (1 mentions)
Rhodopsin, by Abcam (1 mentions)
Heme oxygenase 1, by Cell Signaling Technology (1 mentions)
2 nrf2, by Santa Cruz Biotechnology (1 mentions)
Poly adp ribose polymerase parp, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
2 protocols using protein extraction solution
1
Protein Expression Analysis in Eye
2
Western Blot Analysis of Metallomacrocycle 7
Check if the same lab product or an alternative is used in the 5 most similar protocols
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HepG2 cells (1 × 106 per
60 mm dish) were plated in dishes and allowed to attach for
24 h. After metallomacrocycle7 treatment, cells were
washed with cold PBS, harvested, and cell pellets were lysed with
the protein extraction solution (iNtRON, Korea) on ice for 1 h. Proteins
were separated by centrifugation at 12 000 rpm for 10 min at
4 °C, and aliquots (40 μg) were separated by 12% sodium
dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and
then transferred onto nitrocellulose membranes (Bio-Rad Laboratories
Inc., Hercules, CA). Nonspecific binding was blocked by incubating
membranes with 5% skim milk in TBST (10 nmol/L tris–HCl (pH
8.0), 150 mmol/L NaCl, and 0.1% Tween 20) for 1 h at room temperature
(rt). Membranes were then incubated with 1:1000 diluted primary antibodies
at 4 °C overnight, probed with horseradish peroxidase-conjugated
secondary antibodies (1:5000), and visualized by densitometry (ECL
system, Amersham, U.K.) according to the manufacturer’s instructions.
60 mm dish) were plated in dishes and allowed to attach for
24 h. After metallomacrocycle
washed with cold PBS, harvested, and cell pellets were lysed with
the protein extraction solution (iNtRON, Korea) on ice for 1 h. Proteins
were separated by centrifugation at 12 000 rpm for 10 min at
4 °C, and aliquots (40 μg) were separated by 12% sodium
dodecyl sulfate-polyacrylamide gel (SDS-PAGE) electrophoresis and
then transferred onto nitrocellulose membranes (Bio-Rad Laboratories
Inc., Hercules, CA). Nonspecific binding was blocked by incubating
membranes with 5% skim milk in TBST (10 nmol/L tris–HCl (pH
8.0), 150 mmol/L NaCl, and 0.1% Tween 20) for 1 h at room temperature
(rt). Membranes were then incubated with 1:1000 diluted primary antibodies
at 4 °C overnight, probed with horseradish peroxidase-conjugated
secondary antibodies (1:5000), and visualized by densitometry (ECL
system, Amersham, U.K.) according to the manufacturer’s instructions.
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