The largest database of trusted experimental protocols

2 protocols using sc 73614

1

Protein Expression Analysis of Rat Atrium

Check if the same lab product or an alternative is used in the 5 most similar protocols
Rat atrium tissue lysate and whole cells were prepared by 1x cell lysis (Cell Signaling Technologies, USA) and 1x protease inhibitors (Cat. 04693159001; Roche Molecular Biochemicals, USA). The crude extracts were centrifuged at 13000×g, 4 °C for 10 min and the supernatant was collected. The insoluble sediment was resuspended in 8 mM guanidine hydrochloride in complete lysis. The protein concentration was measured by bicinchoninic acid protein assay and equal amounts of protein samples were separated on 12% polyacrylamide gels, transferred onto PVDF membrane. Membranes were blocked in 5% non-fat milk or 3% BSA at room temperature and incubated with primary antibodies overnight at 4 °C. The primary antibodies targeted against Caspase-3 (Cell Signaling Technology, 9662s), BCL-2 (Abcam, ab32124), BAX (Abcam, ab32503), Vinculin (Santa Cruz Biotechnology, sc-73614), LAMP-2A (Abcam, ab125068), LC3B (Abcam, ab192890), p62 (Abcam, ab109012), MYL4 (Abnova, HOOOO4635-M01), Cathepsin B (Abcam, ab214428) or Cathepsin L (Santa Cruz Biotechnology, sc-390367) at a 1:1000 dilution. After 1-h HRP-conjugated secondary antibodies incubation, bends were visualized using chemiluminescence (ECL, TANON, China) and viewed under Amersham Imager 600 system (GE Healthcare, USA). The relative intensity of each band was normalized to Vinculin.
+ Open protocol
+ Expand
2

Western Blot Analysis of Tau Phosphorylation

Check if the same lab product or an alternative is used in the 5 most similar protocols
Total protein lysates were prepared using NE-PER Kit (Pierce, Rockford, IL, USA) and supplemented with 1X of protease inhibitor cocktail (Roche) according to the manufacturer’s instruction. For detection of proteins 25 μg of total extracts were mixed with denaturing buffer (1× Laemmli loading buffer with 10% of β-mercaptoethanol) and analyzed by SDS–PAGE/Western blot. Separated proteins were transferred onto a 0.45-μm pore size nitrocellulose membrane (Millipore) using the iBlot transfer system (Novex, Thermo Fisher, USA). Membranes were incubated with primary antibodies diluted from 1:500 to 1:1000, followed by TBS-T washes and incubation with HRP (horseradish peroxidase)−conjugated secondary antibodies (GE Healthcare). The signal was visualized by enhanced chemiluminescence with Luminata Forte Western HRP Substrate (Millipore) and the ImageQuant LAS 4000 imaging system. The following antibodies were used: anti-p-TauS262 (Abcam, ab131354), anti-total Tau (Abcam, ab76128), anti-p-Tau-T205 (Santa Cruz biotechnology, sc-73614), anti-VCL (Abcam, ab254410) and anti-p-Tau Y18 (GeneTex, GTX54658), Anti-CFL-1 (Abcam, ab42824). Immunoblot uncut membranes are provided in DATA S1.
+ 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!