The largest database of trusted experimental protocols

2 protocols using horseradishperoxidase conjugated anti rabbit or mouse igg antibody

1

Protein Extraction and Western Blot Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
Total cellular protein was extracted by suspending the cells in sodium dodecyl sulphate (SDS) lysis buffer together with freshly added proteinase inhibitor cocktail (Nacalai tesque, Kyoto, Japan). Lysates were incubated on ice for 30 min with intermittent mixing and then centrifuged at 12,000 rpm for 10 min at 4 °C in an Eppendorf centrifuge. The supernatant was recovered and the protein concentration was determined using the Micro BCA Protein Assay Kit (The Thermo Scientific Pierce, Rockford, IL). Western blot was performed using the enhanced chemiluminescence system. Briefly, extracted cellular proteins were separated by 10% SDS polyacrylamide gels and electrotransferred onto polyvinylidine difluoride membranes. After blocking with 3% bovine serum albumin in PBS, the membranes were incubated with primary antibody for 1.5 h at room temperature or at 4 °C overnight. After washing, the membranes were probed with horseradishperoxidase-conjugated anti-rabbit or -mouse IgG antibody (Cell Signaling, Beverly, MA), and the bands were visualized by using the enhanced chemiluminescence system (Nacalai Tesque, Kyoto, Japan). The chemiluminescent signal was captured with a Fujifilm luminescent image LAS-1000 analyzer (Fujifilm, Tokyo, Japan) and quantified with the Image J software (http://rsb.info.nih.gov/ij). To confirm equal loading of proteins, the membranes were probed for β-actin.
+ Open protocol
+ Expand
2

Synaptic Protein Expression Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols
After the behavioral tests, on P7, P14, or P30, mice were anesthetized with isoflurane, ACC tissues were separated and quickly put into RIPA buffer (BiYunTian, China) consisting of protease and phosphatase inhibitors (Roche, CH). The protein concentration of all of the samples was 2 μg/μL, measured using the BCA Protein Assay Kit (Thermo, United States). The process has been previously described (Qi et al., 2018 (link)). The membranes were incubated with horseradish peroxidase-conjugated anti-rabbit or mouse IgG antibody (1:1000, Cell Signaling Technology) for 1 h at room temperature. The primary antibodies were as follows: VGLUT1 (1:1000, Millipore), PSD95 (1:1000, Abcam), VGAT (1:1000, Synaptic Systems), gephyrin (1:1000, Synaptic Systems), BDNF (1:1000, Abcam), GAPDH (1:1000, Proteintech) and β-actin (1:1000, Proteintech). The images were scanned by a chemiluminescent imaging system (5200 Multi, Tanon, China or UVP ChemStudio PLUS, Analytik Jena, Germany) and were quantified with ImageJ software (Version 1.48). GAPDH and β-actin served as the staining standard.
+ Open protocol
+ Expand

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required

Sign up now

Revolutionizing how scientists
search and build protocols!