At 1 and 4 weeks after transplantation, the cells and rat brain tissue were homogenized in ice-cold RIPA lysis buffer (Solarbio, Beijing, China) supplemented with 1% phenylmethylsulfonyl fluoride (Solarbio), and then centrifuged at 12,000 × g for 3 minutes at 4°C to collect the supernatant according to the manufacturer’s instructions. Protein concentrations were assessed using the bicinchoninic acid (Tiangen Biotech Co., Ltd., Beijing, China) assay. Protein was boiled for 20 minutes before electrophoresis was performed on 8% sodium dodecyl sulfate-polyacrylamide gels. The membranes were blocked with 5% non-fat milk and incubated at 4°C overnight with primary antibodies as follows: rabbit monoclonal anti-CXCR4 (1:800; Abcam), rabbit polyclonal anti-SDF-1 (1:800; Abcam), and rabbit polyclonal anti-β-actin (1:1000; Proteintech). The membranes were subsequently incubated for 1.5 hours with horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit IgG, 1:8000; Proteintech) at room temperature. β-Actin was used as a loading control. Proteins were detected using enhanced chemiluminescent reagent (Millipore, Boston, MA, USA). The relative levels of immunoreactive protein were quantified using ImageJ software (NIH, Bethesda, MD, USA), and the data were normalized to β-actin before statistical analysis.
Bicinchoninic acid (bca)
Manufactured by Tiangen Biotech
Sourced in China
The BCA (Bicinchoninic Acid) is a colorimetric assay used for the quantitative determination of total protein concentration. It is a sensitive and accurate method that utilizes the reduction of Cu2+ to Cu+ by protein in an alkaline medium, and the subsequent chelation of the Cu+ ion with bicinchoninic acid to produce a purple-colored complex that absorbs light at 562 nm.
Automatically generated - may contain errors
Lab products found in correlation
Rabbit monoclonal anti cxcr4, by Abcam (1 mentions)
Enhanced chemiluminescent reagent, by Merck Group (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Phenylmethylsulfonyl fluoride, by Solarbio (1 mentions)
Ab207802, by Abcam (1 mentions)
Ab109411, by Abcam (1 mentions)
Ab283684, by Abcam (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Ab224368, by Abcam (1 mentions)
Ab13556, by Abcam (1 mentions)
Pbs buffer, by Solarbio (1 mentions)
Bovine serum albumin (bsa), by Solarbio (1 mentions)
Bovine serum albumin (bsa), by Maclin Biochemical Technology (1 mentions)
Oca25, by Dataphysics (1 mentions)
Anti vimentin, by Abcam (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti mzf 1, by Santa Cruz Biotechnology (1 mentions)
Anti slug, by Abcam (1 mentions)
Chemiluminescence agent, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by ZSGB-BIO (1 mentions)
5 protocols using bicinchoninic acid (bca)
1
CXCR4 and SDF-1 Expression in Transplanted Cells
2
Protein Expression Analysis in Traumatic Brain Injury
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Proteins were extracted from the pericontusive cortex (width, 3 mm; depth, 3 mm; length, 5 mm) and quantified using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting was performed to quantify the expression of OX1R, TLR4, NF-p65, NLRP3, ASC, and Caspase-1. Bicinchoninic acid (Cat. no. PA115; Tiangen Biotech, China) was used to measure protein concentrations in the tissue lysates. The primary antibodies used for western blotting included anti-OX1R (1:1000, ab224368, Abcam, UK), anti-TLR4 (1:1000, ab13556, Abcam), anti-p65 (1:1000, D14E12, Cell Signaling Technology, USA), anti-NLRP3 (1:500, ERP23094-1, Abcam), anti-caspase-1 (1:500, ab207802, Abcam), anti-ASC (1:1000, ab283684, Abcam), anti-GAPDH (1:2000, 60004-1-lg, Proteintech, China), and anti-HDAC1 (1:2000, ab109411, Abcam). Subsequently, proteins were electrophoretically separated on sodium dodecyl sulfate-polyacrylamide gels and transferred on to polyvinylidene fluoride membranes. Secondary antibodies (1:5000, Zsgb-Bio, China) conjugated to horseradish peroxidase were incubated with the membranes for 60 min at ambient temperature to facilitate primary antibody binding. After western blotting, the Image Lab Software (version 3.0) was used to evaluate the results.
3
Alginate-Polysaccharide Hydrogel for Biomedical Applications
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Sodium alginate (SA, AR, Shanghai Aladdin Biochemical Technology Co., Ltd), atractylodis macrocephalae polysaccharide (laboratory extraction, 92%),22 (link) bovine serum albumin (BSA, biotechnology grade, 96%), 10 × PBS buffer (0.01 M, pH 7.2–7.4, Beijing Solarbio Science & Technology Co., Ltd), BCA (Tiangen Biotech (Beijing) Co, Ltd), Ca(NO3)2 (AR, Tianjin Kaitong Chemical Reagent Co., Ltd).
4
Protein Adsorption on Biocompatible Cages
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The contact angle of water and diiodomethane were assessed by a contact angle tester (OCA25, Dataphysics, Germany), then the surface energy was calculated by the method of Owens two-liquid [8 (link)].
The adherent protein was evaluated by bicinchoninic acid protein assay kit (BCA, TIANGEN, China). The cages (G-PEEK, G-PEEK/Ta-5, G-PEEK/Ta-10 and G-PEEK/Ta-15) were covered by the protein solutions (including 30 μg/mL fibronectin (Fn, Macklin, China) and 5 mg/mL bovine serum albumin (BSA, Macklin, China)) in 12-well plates. After being incubated 4 h at 37 °C, the unabsorbed proteins were cleaned with PBS 3 times. Finally, under shaking at 37 °C, the cages were covered by 5% sodium dodecyl sulfate (SDS) to thoroughly set free the absorbed proteins, then were evaluated by the BCA assay kit.
The adherent protein was evaluated by bicinchoninic acid protein assay kit (BCA, TIANGEN, China). The cages (G-PEEK, G-PEEK/Ta-5, G-PEEK/Ta-10 and G-PEEK/Ta-15) were covered by the protein solutions (including 30 μg/mL fibronectin (Fn, Macklin, China) and 5 mg/mL bovine serum albumin (BSA, Macklin, China)) in 12-well plates. After being incubated 4 h at 37 °C, the unabsorbed proteins were cleaned with PBS 3 times. Finally, under shaking at 37 °C, the cages were covered by 5% sodium dodecyl sulfate (SDS) to thoroughly set free the absorbed proteins, then were evaluated by the BCA assay kit.
5
Western Blot Analysis of EMT Markers
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The Western bolt analysis was performed as described previously [18 (link)]. The protein concentrations of cell or tissue lysates were measured using the Bicinchoninic Acid Assay (BCA) (TIANGEN, Beijing, China, PA115). Afterward, 60 µg protein was separated using 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroblotted onto a nitrocellulose membrane. The membranes were blocked with 5% non-fat milk for two hours at room temperature, immunoblotted with specific primary antibodies. The antibodies used in this experiment were as follows: anti-E-cadherin (CST, Boston, MA, USA, 3195S; 1:1, 000); anti-N-cadherin (CST, Boston, MA, USA, 13116S, 1:1, 000); anti-Vimentin (Abcam, Cambridge, UK, ab8978, 1:1, 000); anti-Slug (Abcam, Cambridge, UK, ab51772, 1:1, 000); anti-β-actin (ZSGB-BIO, Beijing, China; TA-09, 1:1, 000); anti-GAPDH (Santa Cruz, CA, USA, sc-25778, 1:1, 000); anti-MZF1 (Santa Cruz, CA, USA, 293218, 1:1, 000). All antibodies were used following the manufacturer’s instructions. Protein expression was detected using a chemiluminescence agent (Thermo, Waltham, MA, USA). ImageJ software (Bethesda, MD, USA) was employed to quantify the results.
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