Total protein of the brain or cells was extracted using RIPA Lysis Buffer (P0013B, Beyotime Biotechnology, China) following the instructions in the manual. After quantifying the proteins with the BCA method, samples were denatured via boiling for 5 min. Following electrophoresis, transmembrane, and blocking, the target proteins were labeled with primary antibodies: β-actin (1:5000, rabbit polyclonal, Proteintech, 20536-1-AP), BDNF (1:1000, rabbit polyclonal, Proteintech, 28205-1-AP), TrkB (1:1000, rabbit polyclonal, Proteintech, 13129-1-AP), p75NTR (1:1000, rabbit polyclonal, Proteintech, 55014-1-AP), pCREB (Ser133, 1:1000, rabbit polyclonal, Affinity, AF3189), pTrkB (Tyr706, 1:1000, rabbit polyclonal, Affinity, AF3462). HRP Conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (1:5000, Boster, BA1054) was used to amplify the signal of the target proteins. Protein bands were visualized using the Enhanced ECL Chemiluminescent Substrate Kit (Millipore, United States) and imaged using a chemiluminescence instrument (Bio-Rad, United States). Quantitative analysis was performed by measuring the band intensities using ImageJ software (NIH, United States).
Enhanced ecl chemiluminescent substrate kit
Manufactured by Merck Group
Sourced in United States
The Enhanced ECL Chemiluminescent Substrate Kit is a laboratory product designed to detect and quantify proteins in Western blot analyses. It utilizes a chemiluminescent reaction to generate a luminescent signal that can be captured and measured using appropriate detection equipment.
Automatically generated - may contain errors
Lab products found in correlation
Chemiluminescence instrument, by Bio-Rad (1 mentions)
Hrp conjugated affinipure goat anti rabbit igg h l, by Boster Bio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
β actin, by Proteintech (1 mentions)
Ripa lysis buffer, by Takara Bio (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
2 protocols using enhanced ecl chemiluminescent substrate kit
1
Quantitative Analysis of Brain Proteins
2
Exosome Protein Characterization Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Total protein of HNPCs or bMSCs-derived exosomes was obtained using RIPA lysis buffer (Takara). Approximately 30 μg protein of each group were separated by 12 % SDS-PAGE and transferred onto PVDF membranes (Millipore, USA). After blocking by 5 % skim milk for 1 h at RT, the membranes were incubated with primary antibody against U2AF2 (1:1000, #15624-1-AP, Proteintech, China), ACAN (1:1000, #bs-1223 R, Bioss, China), Collagen I (1:1000, #bs-7158 R, Bioss), Collagen II (1:1000, #bs-10589 R, Bioss), CD63 (1:1000, #bs-1523 R, Bioss), CD81 (1:1000, #bs-6934 R, Bioss) or internal reference GAPDH (1:10000, #KC-5G5, Aksomics, China) overnight at 4 °C, respectively. On the next day, the membranes were then incubated with secondary antibody for 1hr at RT. The target protein bands were visualized by an Enhanced ECL Chemiluminescent Substrate Kit (Millipore) and the relative gray values were analyzed by Image J (NIH, USA).
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