Filtered (centrifree ultrafiltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-N4-250/CF, R&D Systems, Canada) of known concentrations (typically 250 mgL−1) in the phosphate buffered saline (PBS) pH 7.4 ± 0.2, 0.15 M (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration in the solvent. To minimize errors in concentration measurements, two complementary spectrophotometric techniques were used: the BCA (protein quantification bicinchoninic acid assay, kit for low concentration, ABCAm, Cambridge, UK) and UV absorbance at 280 nm measured with microplate spectrophotometer (BioTek Epoch, United States). Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01–2 mgL−1. The exact concentration of these solutions after membrane filtration was determined by commercially available Enzyme-Linked Immunosorbent Assay (ELISA) (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298 ± 0.1 K.
Bicinchoninic acid (bca)
Manufactured by Abcam
Sourced in United Kingdom, United States
The BCA (Bicinchoninic Acid) is a colorimetric assay used for the quantitative determination of total protein concentration. It combines the well-known reduction of Cu2+ to Cu+ by protein in an alkaline medium with the highly sensitive and selective colorimetric detection of the cuprous cation (Cu+) by bicinchoninic acid.
Automatically generated - may contain errors
Lab products found in correlation
Dy999, by R&D Systems (2 mentions)
Dy994, by R&D Systems (2 mentions)
Wa126, by R&D Systems (2 mentions)
Dy995, by R&D Systems (2 mentions)
Dy268, by R&D Systems (2 mentions)
248 n4 250 cf, by R&D Systems (2 mentions)
Dy992, by R&D Systems (2 mentions)
Dy006, by R&D Systems (2 mentions)
Dy990, by R&D Systems (2 mentions)
Ripa lysis buffer, by Ipsen (1 mentions)
Goat anti mouse igg, by Beyotime (1 mentions)
Goat anti rabbit igg, by Proteintech (1 mentions)
Protein assay kit, by Abcam (1 mentions)
Enhanced chemiluminescence, by Beyotime (1 mentions)
Minute total protein extraction kit, by Invent Biotechnologies (1 mentions)
Gpx4 antibody, by Abcam (1 mentions)
Sds page, by Merck Group (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Lc3 2, by Abcam (1 mentions)
Hrp labeled secondary antibody, by Abcam (1 mentions)
5 protocols using bicinchoninic acid (bca)
1
Quantification of Recombinant BDNF
2
Western Blot Analysis of Cell Proteins
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Proteins of cells and tissues were extracted with RIPA Lysis Buffer (EpiZyme, Shanghai, China), and quantified with bicinchoninic acid (BCA, ABCAm, Cambridge, MA, USA). Equal amounts of protein (25μg/lane) were separated by electrophoresis, and then transferred into membranes with a pore size of 0.2μm (Merck Millipore, Billerica, MA, USA). The membranes were blocked, and then incubated with the following antibodies: ABHD6 rabbit antibody (#97573, 1:1000, Cell Signaling TECHNOLOGY, Danvers, MA, USA), CD36 goat antibody (#Q3UAI3, 1:2000, R&D Systems, Minneapolis, MA, USA), PPARγ mouse antibody (#sc-7273, 1:500, Santa Cruz, CA, USA), GLP-1R rabbit antibody (#26196-1-AP, 1:500, Proteintech, Wuhan, HB, China) or β-actin mouse antibody (#AF0003, 1:5000, Beyotime, Shanghai, China). Secondary antibodies were as follows: horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (#A0208, 1:1000, Beyotime, Shanghai, China), goat anti-mouse IgG (#A0216, 1:1000, Beyotime, Shanghai, China) or rabbit anti-goat IgG (#SA00001-4, 1:2000, Proteintech, Wuhan, HB, China). Bands were detected using an enhanced chemiluminescence assay (ECL, NCM Biotech, Suzhou, JS, China) according to the manufacturer’s instructions. The representative blot bands were repeated at least three times.
3
Protein Extraction and Western Blot Analysis in KGN Cells and PCOS Mice
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Total protein of KGN cells and PCOS mice was extracted by using Minute™ total protein extraction kit (Invent, Eden Prairie, MN, USA), and the concentration of protein was detected by BCA (bicinchoninic acid) protein assay kit (ABCAm). The primary antibodies were SKP2 antibody (1:500, ABCAm, ab183039), NEDD4L antibody (1:500, ABCAm, ab168349), GPX4 antibody (1:500, ABCAm, ab134953), UB antibody, and anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (1:5000, ABCAm, ab9485). The membranes were washed with TBST (TBS with Tween-20) for three times after incubation with primary antibodies for 2 h. Thereafter, membranes were incubated with goat anti-rabbit IgG H&L (HRP) (1:5000, ab205718) for 1 h at 37°C. The signals were visualized using enhanced chemiluminescence (Beyotime, Shanghai, China).
4
Quantifying Autophagy Proteins by Western Blot
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BCA (ABCAm) was utilized for protein quantification after RIPA lysis (ABCAm) of the cells. The proteins were then transferred to a PVDF (Sigma-Aldrich) membrane via SDS-PAGE (Sigma-Aldrich), sealed with 5 % skim milk for 2h, and added with LC3-II, Beclin-1 and GAPDH primary antibodies (1:1,000, ABCAm). After incubation at 4 ℃ overnight, an HRP labeled secondary antibody (1:5,000, ABCAm) was added to incubate at an ambient temperature for 1h, followed by development using ECL, and gray value analysis with Image J.
5
BDNF Protein Standardization Protocol
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Filtered (centrifree ultra ltration device, Merck Group, Darmstadt, Germany) stock solutions of carrier free recombinant human BDNF (248-N4-250/CF, R&D Systems, Canada) of known concentrations (typically 250 mgL -1 ) in the phosphate buffered saline (PBS) pH 7.4 +/-0.2, 0.15M (Biomed, Lublin, Poland) were prepared to remove aggregates and provide constant, free form protein molecules concentration in the solvent. To minimize errors in concentration measurements, two complementary spectrophotometric techniques were used: the BCA (protein quanti cation bicinchoninic acid assay, kit for low concentration, ABCAm, Cambridge, UK) and UV absorbance at 280 nm measured with microplate spectrophotometer (BioTek Epoch, United States). Prior to each measurement, the stock solution was diluted to a desired bulk concentration, typically 0.01-2 mgL -1 . The exact concentration of these solutions after membrane ltration was determined by commercially available Enzyme-Linked Immunosorbent Assay (ELISA) (DY992, DY990, DY994, DY999, DY995, WA126, DY006, DY268, R&D Systems). The temperature of experiments was kept at a constant value equal to 298±0.1K.
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