Quick starttm bradford protein assay kit

Initially, brain tissue samples were weighed and homogenized in cryogenic mill (Freezer Mill 6770—Spex Sample Preparation, Metuchen, NJ, USA), and cryofractured at low temperatures (liquid nitrogen). Then, tissue samples (50 μg) were added to 500 μL of lysis buffer A (Urea/Thiourea), incubated for 2:30 h in refrigerator with constant agitation, following centrifugation at 14,000 rpm for 30 min at 4 °C. Supernatant was collected and submitted to Bradford assay for total protein quantification, using the Quick Start TM Bradford Protein Assay kit (Bio-rad, Hercules, CA, USA), in duplicate, as described previously [45 (link)]. For each reaction, 5 μL of sample plus 250 μL of staining reagent was used. After incubation for 5 min at room temperature, absorbances were determined at 595 nm in spectrophotometer. Protein concentrations were calculated by comparing with a standard curve containing Bradford concentrations.

Cytoplasmic and nuclear protein extracts were then prepared using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Thermo Scientific, Waltham, MA, USA) following the manufacturer’s protocol, as described elsewhere [64 (link)]. The quantification of the protein extracts was carried out using the Quick Start^TM Bradford Protein Assay Kit (Bio-Rad), and the absorbances were measured using Ascent Multiskan at 590 nm.
For Western blot analysis, 60 μg of protein lysate was loaded into each well of a gel and size-fractionated using 12% SDS-PAGE under reducing conditions. The proteins were transferred onto a nitrocellulose membrane using Trans-Blot^® Turbo^TM Transfer Systems RTA Transfer Kits (Bio-Rad) at 25 V for 30 min. The membrane was then blocked with 5% (w/v) skim milk in Tris saline at 4 °C overnight. Subsequently, the membrane was incubated with a primary antibody specific to the total and phosphorylated rabbit anti-human MEK1/2 and PKC (Cell Signalling Technology, Danvers, MA, USA). The blot was washed three times with Tri-buffered saline with Tween-20 (TBST) and incubated with anti-rabbit IgG-conjugated with HRP secondary antibody (Cell Signaling). Finally, the proteins were detected using the DAB Substrate Kit following the manufacturer’s instructions.

His-tagged maltose-binding protein (MBP) was expressed from a plasmid in E. coli and then purified through Ni-chelated Sepharose Fast Flow Resin (GE Healthcare) and HiLoad 16/60 Superdex 75 gel filtration column (GE Healthcare). The protein was exchanged into assay buffer (120 mM NaCl, 20 mM NaH₂PO₄/Na₂HPO₄, pH 7.4) by dialysis. Both carbonic anhydrases were obtained from a commercial vendor (h-CA I (Sigma C4396) and b-CA II (Sigma C2522)). All protein concentrations were determined with Quick Start^TM Bradford Protein Assay Kit (Bio-Rad, catalog no. 5000201).
Ligands were all obtained from commercial vendors, as follows: maltose (EMD Millipore 105910), acetazolamide (Sigma 97582), methazolamide (Sigma SML0720), sulfanilamide (Sigma 46874), trifluoromethanesulfonamide (Sigma 638455), and 4-nitrophenyl acetate (Sigma N8130).

Transfected cells were trypsinized and washed with ice-cold 1X PBS in pre-cooled microcentrifuge tubes and lysed on ice using Mammalian Protein Extraction Reagent (M-PER^TM, Cat#78503, ThermoFisher Scientific, Waltham, MA, USA) containing 1% proteinase inhibitor cocktail (Cat#4693159001, Millipore Sigma, Burlington, MA, USA) for 1 h on ice with gentle shaking. The microcentrifuge tube containing the lysate was centrifuged at 15,000 rpm at 4 °C for 35 min to pellet the cellular debris. The supernatant was transferred into a chilled microcentrifuge tube and the pellet was discarded. The protein concentration was determined using a Quick Start^TM Bradford Protein Assay kit (Cat#5000201, Bio-Rad, Hercules, CA, USA), and bovine serum albumin (BSA) was used as the protein standard. The lysates were then frozen at −80 °C for protein analysis using SDS-PAGE.

Escherichia coli DH5α (Invitrogen, Schwerte, Germany) was used for cloning, and E. coli BL21 (DE3) (Invitrogen, Schwerte, Germany) was used for protein expression. pET26b (+) vector was purchased from Novagen (Darmstadt, Germany).
Luria-Bertani (LB) media consisted of: 0.5% (w/v) NaCl, 1% (w/v) peptone, and 0.5% (w/v) yeast extract, and sterilized by autoclaving. Agar was added for preparation of standard agar media at 1.5% (w/v). Lysis buffer consisted of: 50 mM NaH₂PO₄, 300 mM NaCl, and 20 mM imidazole, pH 7.8.
Nco1 and Xho1 restriction enzymes, shrimp alkaline phosphatase, and phusion high-fidelity DNA polymerase were purchased from New England Biolabs (Massachusetts, USA). Trizol^® reagent, T4 DNA ligase, Thermo Scientific RevertAid H Minus first strand cDNA synthesis kit, and PageRuler unstained protein ladder were purchased from Thermo Fisher Scientific (Ohio, USA). Zyppy^TM plasmid miniprep kit, used for plasmid isolation, Zymoclean^TM Gel DNA recovery kit, used for DNA purification, and Mix and Go E. coli transformation kit were purchased from Zymo Research (Irvine, California, USA). HyperLadder^TM 1 kb was purchased from Bioline (London, UK). Protein concentration was determined using Quick Start^TM Bradford protein assay kit from Bio-Rad (Washington, USA).

The infected or transfected cells were trypsinized and washed with ice-cold 1X PBS in pre-cooled microcentrifuge tubes and lysed with ice-cold lysis buffer (Cat#78503, ThermoFisher Scientific, Waltham, MA, USA) containing 1% protease inhibitor (0.5 mL per 5 × 10⁶ cells in 60 mm dish or 75 cm² flask) for 2 h at room temperature or 4 °C overnight with gentle shaking. The microcentrifuge tube containing the lysate was centrifuged at 14,000 rpm at 4 °C for 45 min to pellet the cellular debris. The supernatant was transferred into a clean chilled microcentrifuge tube, kept on ice, and the pellet was discarded. The protein concentration was determined using a Quick Start^TM Bradford Protein Assay kit (Cat#5000201, Bio-Rad, Hercules, CA, USA), and bovine serum albumin (BSA) was used as the protein standard. The lysates were then frozen at −80 °C for protein analysis using SDS-PAGE.

As a basic step, we determined the total protein concentration in 5-µL samples of the kidney homogenates. We used a Bio-Rad Quick StartTM Bradford Protein Assay kit (Hercules, western Contra Costa, CA, USA). Equal protein amounts from the kidney homogenates were denaturated using 4× Laemmli sample buffer from Bio-Rad, and then subjected to SDS polyacrylamide gel electrophoresis for separation of the proteins. Then, we transferred the proteins to a nitrocellulose membrane and blocked the free sites on the membranes by incubation for 1 h in Bio-Rad non-fat dried milk. After that, we washed the membranes and incubated them with primary antibodies for p-p53 (ab33889), smad2/3 (202445), and β-actin (Ab8226) (Abcam, Waltham, MA, USA) overnight. Then, we washed the blots and added secondary antibodies conjugated with horseradish peroxidase. An Amersham BioSciences chemiluminescence kit (Buckinghamshire, UK) was used to detect the concentration of each protein. Then, the blots were imaged, and the band densities were measured by ImageJ software (NIH, Bethesda, MD, USA).