Five micrograms of protein were electrophoresed in 10% SDS-PAGE gels and blotted to polyvinylidine difluoride membranes. Specific primary antibodies (ProteinTech Group Inc.) were detected with peroxidase-labeled secondary antibodies (Amersham, Piscataway, NJ) using SuperSignal West Dura Extended Duration Substrate (Pierce Chemical) per manufacturer's instructions.
Specific primary antibodies
Manufactured by Proteintech
Sourced in United States
Proteintech's specific primary antibodies are high-quality reagents used in various immunoassays to detect and quantify target proteins. These antibodies are developed and validated to provide reliable and reproducible results.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (2 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Peroxidase labeled secondary antibodies, by Cytiva (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
Ecl kit, by Santa Cruz Biotechnology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence ecl reagent, by Thermo Fisher Scientific (1 mentions)
Bca protein kit, by Beyotime (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
0.45 m polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Proteinase inhibitor cocktail, by Beyotime (1 mentions)
Phosphatase inhibitor cocktail, by Yeasen (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Neobioscience (1 mentions)
4 protocols using specific primary antibodies
1
Protein Detection by Western Blotting
2
Western Blot Analysis of Protein Expression
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Total protein was extracted from transfected cells using the mammalian protein extraction reagent RIPA (Beyotime, China). Total proteins from the lysates were separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, USA). The membranes were blocked in 5% nonfat milk and then incubated with diluted specific primary antibodies against TAZ (Proteintech, USA) and ATCB (Proteintech, USA) overnight at 4°C. Primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies. The bands were visualized using an enhanced chemiluminescent kit (ECL kit, Santa Cruz, USA) according to the manufacturer’s protocol.
3
Western Blot Analysis of Neuronal Proteins
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Protein levels of GLP-1R, Akt, p-Akt (T308), and cleaved-caspase-3 were detected by Western blot, with GAPDH as internal reference protein following previous studies [19 (link),20 (link)]. NPCs were lysed using the RIPA lysis buffer (Beyotime, China) on the ice for 20 min and the protein concentration was quantified with a Protein BCA Kit (Beyotime, China). Equal amounts of protein samples were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to the PVDF membrane (Merck Millipore, Billerica, MA, U.S.A.). The membranes were blocked with 5% non-fat milk in TBST (50 mmol/l Tris, pH 7.6, 150 mmol/l NaCl, 0.1%) for 1 h at the room temperature and incubated with specific primary antibodies (Proteintech, Wuhan, China) at 4°C overnight. After washing three times in TBST, membranes were incubated with the secondary antibodies (Proteintech, Wuhan, China) for 2 h at 37°C. The enhanced chemiluminescence (ECL) reagent (Thermo, U.S.A.) was used to detecte protein bands, and grey values were analysed using ImageJ software (National Institutes of Health, U.S.A.).
4
Protein Extraction and Western Blot Analysis
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Cells were washed twice with cold PBS. Total protein was extracted by RIPA lysis buffer accompanied with 1 mM phenylmethylsulfonyl fluoride along with a proteinase inhibitor cocktail (Beyotime) and phosphatase inhibitor cocktail (Yeasen) and then quantified using the BCA Protein Assay kit (Beyotime). Western blotting assay was performed as described.55 (link) Extracts of soluble cellular protein (20 µg) were separated by 10% SDS-PAGE and then transferred to a 0.45 µm polyvinylidene difluoride membrane (Millipore). After blocking with 5% bovine serum albumin for 1 h at 25 °C, each membrane was incubated with specific primary antibodies (Proteintech or Cell Signaling Technology) at 4 °C overnight. On the next day, each membrane was incubated with an appropriate horseradish peroxidase-conjugated secondary antibody (Neobioscience) for 1 h at room temperature. Immunopositive bands were visualized via chemiluminescence (ECL reagent, Millipore) using a Model 4600 Chemiluminescence Imaging System (Tanon).
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