Western blotting was performed as previously described [26 (link)]. Proteins from the brain samples and cultured HT22 cells were lysed with RIPA lysis buffer. Equal amounts of protein (50 μg) were loaded onto a sodium dodecyl sulfate–polyacrylamide gel. The proteins were electrophoresed until they were fully separated and then transferred to a polyvinylidene difluoride membrane. The membrane was then blocked with 5% bovine serum albumin for 1 h at room temperature. The membranes were incubated with the following primary antibodies overnight at 4 ℃: anti-CCR2 (#12199, 1:1000, CST), anti-p-PI3K (#4228, 1:1000, CST), anti-PI3K (#AF7742, 1:1000, Beyotime), anti-AKT (#4691, 1:1000, CST), anti-pAKT (#4060, 1:1000, CST), anti-IL-1β (#ab254360, 1:1000, Abcam), anti-TNF-α (#ab255275, 1:1000, Abcam), anti-BAX (#GB114122, 1:1000, ServiceBio), anti-Bcl-2 (#GB113375, 1:1000, ServiceBio), anti-cleaved caspase 3 (#ab2302, CC3, 1:1000, Abcam), and anti-β-tubulin (#GB11017, 1:1000, ServiceBio). Suitable secondary antibodies (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) were selected and incubated with the membrane at room temperature for 1 h. The bands were then observed using an enhanced chemiluminescence reagent. Image J software (NIH, Bethesda, MD, USA) was used for density measurements and quantification.
Af7742
Manufactured by Beyotime
Sourced in China
The AF7742 is a laboratory centrifuge designed for general-purpose applications. It features a fixed-angle rotor capable of handling sample volumes up to 100 mL. The centrifuge operates at a maximum speed of 6,000 rpm, providing a maximum relative centrifugal force of 4,500 x g. The device is suitable for a variety of sample separation and preparation tasks in research and clinical laboratories.
Automatically generated - may contain errors
Lab products found in correlation
Ab254360, by Abcam (1 mentions)
Ab255275, by Abcam (1 mentions)
Gb11017, by Wuhan Servicebio Technology (1 mentions)
Gb114122, by Wuhan Servicebio Technology (1 mentions)
Gb113375, by Wuhan Servicebio Technology (1 mentions)
Ab112543, by Abcam (1 mentions)
Af1789, by Beyotime (1 mentions)
Ab9485, by Abcam (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
2 protocols using af7742
1
Western Blot Analysis of Molecular Markers
2
Protein Expression Analysis Workflow
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted, separated, and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA), as previously described [33] . These membranes were then incubated with blocking buffer for an hour and subjected to the appropriate primary antibodies for 24 h at 4°C. The primary antibodies included anti-THBS2 (1:1,000, ab112543; Abcam, Cambridge, UK), anti-GAPDH (1:2,500, ab9485; Abcam), anti-p-PI3K (1:1,000, AF5905; Beyotime, Jiangsu, China), anti-PI3K (1:1,000, AF7742; Beyotime), anti-p-AKT (1:1,000, AF1546; Beyotime), and anti-AKT (1:1,000, AF1789; Beyotime). Next, these membranes were probed with horseradish peroxidase-labeled secondary antibodies for 1 h at room temperature. Protein blots were developed using the enhanced chemiluminescence method (Invitrogen). Protein intensity was scanned by using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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
Revolutionizing how scientists
search and build protocols!