Protein lysates were collected on ice in RIPA buffer containing protease and phosphatase inhibitors following a PBS wash. Lysates were spun at 16,000 RCF and the supernatants were collected and used for further analysis. Protein lysates (12.5 μg/lane) were separated using precast NuPAGE™ 4–12% Bis-Tris Protein gels followed by transfer to Immobilon-FL membranes, and blocked with 10% SeaBlock solution. Primary antibodies were used at dilutions of 1:1,000 with 16 h incubations at 4μ, and secondary antibodies were used at dilutions of 1:2,500 with 90 min incubations at 20°C. Images were captured using the LiCoR Odyssey. Primary antibodies were purchased from Cell Signaling: anti-phospho-FoxO1Ser256 (# 9461), anti-FoxO1 (# 14952), anti-phospho-PRAS40Thr246 (# 2997), anti-PRAS40 (# 2691), anti-phospho-AktSer473 (# 4060), anti-Akt (# 4691). Secondary antibodies included IR800- conjugated goat anti-mouse IgG (Rockland, Gilbertsville, PA) and goat anti-rabbit IgG conjugated to Alexa Fluor 680 (Invitrogen, Carlsbad, CA).
Anti phospho pras40thr246
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-phospho-PRAS40Thr246 is a laboratory reagent that specifically detects the phosphorylation of the PRAS40 protein on the threonine 246 residue. This antibody can be used in various techniques, such as Western blotting, to analyze the phosphorylation status of PRAS40 in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti pras40, by Cell Signaling Technology (3 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (2 mentions)
Alexa fluor 680, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti phospho foxo1 ser256, by Cell Signaling Technology (1 mentions)
Anti foxo1, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ecl western blot detection kit, by Merck Group (1 mentions)
Infinite m200 pro, by Tecan (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Anti phospho mtor ser2448, by Merck Group (1 mentions)
Anti phospho gsk3β ser9, by Cell Signaling Technology (1 mentions)
Anti phospho akt thr308, by Cell Signaling Technology (1 mentions)
Anti phospho ampkα thr172, by Cell Signaling Technology (1 mentions)
Anti acc, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti gsk3β, by Cell Signaling Technology (1 mentions)
Anti 4e bp1, by Cell Signaling Technology (1 mentions)
Anti phospho s6k thr389, by Cell Signaling Technology (1 mentions)
Anti phospho ikkα β ser176 180, by Cell Signaling Technology (1 mentions)
3 protocols using anti phospho pras40thr246
1
Western Blot Analysis of PI3K/Akt Pathway
2
Protein Isolation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins in PC12 cells were isolated using RIPA Lysis Buffer (Beyotime Biotechnology). The protein concentration was determined using a BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) with a full-wavelength functional microplate reader (Infinite M200Pro, Tecan, Switzerland). Then equal concentration Proteins were separated using 12.5% SDS–PAGE and transferred to nitrocellulose membranes. After blocking in 10% nonfat dry milk for 1 h, phosphorylated proteins were blocked using bovine serum albumin (5% BSA, room temperature) for 2 h. The membranes were incubated overnight at 4°C with the following primary antibodies: anti-phospho-mTOR-(Ser2448) (ZRB1553), anti-m-TOR(SAB5700687), anti-LC3 (SAB5701328) (Sigma Aldrich, United States), anti-phospho-PRAS40-(Thr246) (Cell Signaling) and anti-PRAS40(Cell Signaling)antibodies. The membrane was then incubated with the corresponding secondary antibody for 1 h. After three washes with PBST, the membrane was visualized using an ECL Western blot detection kit (Merck, United States). The average optical density of the images was analyzed using ImageJ.
3
Signaling Pathway Antibody Panel Protocol
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!