The total protein of cells was homogenized in cold RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing protease inhibitor (100×) and phosphatase inhibitor (Sangon Biotech Inc., Shanghai, China). The protein concentration was determined using the BCA kit according to the introduction. SDS-PAGE (BIO-RAD Co., California, USA) was used to separate protein by electrophoresis. After that, the protein was transferred to the nitrocellulose membrane (Pall Co., NY, USA). We indicated the different range of membrane cutting according to PageRuler Prestained Protein Ladder (10–170 kDa; Thermo, #26616). The membrane with phosphorylated protein was blocked with bovine serum albumin (Sangon Biotech Inc., Shanghai, China), while that with nonphosphorylated protein was blocked with 5% nonfat dried milk (Sangon Biotech Inc., Shanghai, China) for 1 h. Then, the membrane was cultured with primary antibodies overnight at 4°C. The membrane was placed in secondary antibodies for 1 h at room temperature. After that, ECL luminescence solution was added, and the gray value of the bands was automatically developed and read by Molecular Imager Chemi Doc XRS (BIO-RAD Co., California, USA) and JS-780 automatic gel imaging analysis systems. GAPDH was used as the internal control.
Nonfat dried milk
Manufactured by Sangon
Sourced in China
5% nonfat dried milk is a laboratory reagent used as a blocking agent in various immunoassay techniques. It serves to reduce non-specific binding and background signals during the detection process.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Sangon (2 mentions)
Phosphatase inhibitor, by Sangon (1 mentions)
Pageruler prestained protein ladder, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Molecular imager chemidoc xrs, by Bio-Rad (1 mentions)
Sds page, by Bio-Rad (1 mentions)
Protease inhibitor, by Sangon (1 mentions)
Irdye 800cw goat anti rabbit antibody, by LI COR (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Odyssey infrared imaging system, by LI COR (1 mentions)
Ripa lysis buffer, by Sangon (1 mentions)
Antibody against nrf2, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Sangon (1 mentions)
Loading buffer, by Sangon (1 mentions)
Gsh px, by Nanjing Jiancheng (1 mentions)
Bca kit, by Sangon (1 mentions)
Temed, by Sangon (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
4 protocols using nonfat dried milk
1
Protein Extraction and Western Blotting
2
Quantitative Protein Analysis in Transplantation Studies
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Protein was isolated from the cells and brain tissues from the transplantation sites with TRIzol (Invitrogen) according to the manufacturer's manual. Protein concentrations were tested by a bicinchoninic acid assay (BCA) protein assay (Pierce, New York, NY, USA). Samples (30 μg protein/lane) were concentrated and separated by SDS-PAGE on 5% and 15% acrylamide gels (Sangon, Shanghai, China) and then transferred to Immun-Blot polyvinylidene difluoride membranes (PVDF; Bio-Rad, Hercules, CA, USA). Membranes were blocked in 5% nonfat dried milk (Sangon) in PBS with 0.1% Tween-20 (PBST) for 1 h at room temperature, followed by incubation with primary antibodies at 4°C overnight. The primary antibodies included polyclonal rabbit b-actin antibody (1:1,000; Abcam, Cambridge, UK), mouse tyrosine hydroxylase (TH) antibody (1:1,000; Sigma-Aldrich), and rabbit glial fibrillary acidic protein (GFAP) antibody (1:800; Sigma-Aldrich). After being washed three times with PBST, the membranes were incubated with goat anti-rabbit IRDye ® 800CW antibody or goat anti-mouse IRDye ® 700CW antibody (Li-Cor Biosciences, Lincoln, NE, USA) diluted 1:10,000 in blocking buffer (1 h) and washed three times. Protein bands were scanned and analyzed through Odyssey Infrared Imaging System (Li-Cor Biosciences) .
3
Seleno-Aminopolysaccharide Antioxidant Pathway
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DAO, D-LA, and LPS kits were obtained from Nanjing Senbeijia Biological Technology Co., Ltd. (Nanjing, China). NO, TNOS, SOD, GSH-Px, CAT, and MDA were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). 2000 DNA Marker, RNA loading buffer, DNA 6×loading buffer, RIPA Lysis buffer, PMSF, BCA kit, BeyoECL Star kit, 5×loading buffer, BSA, non-fat dried milk, TEMED were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Monoclonal antibodies against β-actin were from Cell Signaling Technology, Inc. (Boston, MA, USA). Antibody against Nrf2 was acquired from Abcam (Cambridge, UK). The antibody against Keap1 was acquired from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Alcian Blue, periodic acid schiff were purchased from Beijing solab technology co., Ltd. (Beijing, China). All other reagents are of the highest grade or analytical grade. Our laboratory synthesized the low molecular seleno-aminopolysaccharide (LSA).
4
Western Blot Analysis of FTO Protein
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The tissues of each group were lysed on ice with lysis buffer (Beyotime Institute
of Biotechnology, Shanghai, China) for 10 min. The supernatant was obtained by
centrifugation (1000 ×g) at 4°C for 20 min. After sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (BIO-RAD Co., California, United
States of America), the protein was transferred to nitrocellulose membrane (Pall
Co., New York, United States of America). After being blocked with nonfat dried
milk (Sangon Biotech Inc., Shanghai, China), the membrane was incubated with
anti-FTO antibody anti-β-actin antibody (Abcam Technology, Cambridge,
United Kingdom) overnight at 4°C. The membrane was then incubated with secondary
antibody (ZSGB Biotech Co., Ltd., Beijing, China) for one hour at 25°C. Enhanced
chemiluminescence solution was added to the darkroom for development, exposure,
and photography by gel imager. With β-actin as internal reference, the
data were analyzed by QuantityOne image analysis software.
of Biotechnology, Shanghai, China) for 10 min. The supernatant was obtained by
centrifugation (1000 ×g) at 4°C for 20 min. After sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (BIO-RAD Co., California, United
States of America), the protein was transferred to nitrocellulose membrane (Pall
Co., New York, United States of America). After being blocked with nonfat dried
milk (Sangon Biotech Inc., Shanghai, China), the membrane was incubated with
anti-FTO antibody anti-β-actin antibody (Abcam Technology, Cambridge,
United Kingdom) overnight at 4°C. The membrane was then incubated with secondary
antibody (ZSGB Biotech Co., Ltd., Beijing, China) for one hour at 25°C. Enhanced
chemiluminescence solution was added to the darkroom for development, exposure,
and photography by gel imager. With β-actin as internal reference, the
data were analyzed by QuantityOne image analysis software.
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