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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.

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2 protocols using protein extraction solution

1

Protein Expression Analysis in Eye

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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.
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2

Western Blot Analysis of Metallomacrocycle 7

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HepG2 cells (1 × 106 per
60 mm dish) were plated in dishes and allowed to attach for
24 h. After metallomacrocycle 7 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.
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