Enolase activity was determined by direct spectrophotometric assay via measuring the increase of absorbance at 240 nm of phosphoenolpyruvate (PEP) as described previously with some modifications (54 (link)). Briefly, the reaction buffer (pH 7.0) containing 10 mM imidazole, 200 mM KCl, and 0.5 mM MgAc in a final reaction volume of 100 μL was mixed with Eno1 (a final concentration of 30 nM), followed by the mixture of 2-phosphoglycerate (2-PG) (Yuanye Biotech Shanghai) with a final concentration of 1 mM. The enolase activity of Eno1 was evaluated by measuring the increase of absorbance (OD240) at room temperature for 10 min. For the inhibition study of enolase by BE, different concentrations of BE were used to mix well with Eno1 and were incubated at room temperature for 5 min, and subsequent operations were described above. In the final reaction volume of 100 μL of this assay, the initial concentration of BE was 200 μM and serially diluted 2-fold for eight different concentrations. The well-known enolase inhibitor sodium fluoride (NaF; Sangon Biotech) was initiated from 5 mM and used as a control (29 (link)). Inhibition curves were fitted by nonlinear regression (four parameters) using the GraphPad Prism software.
Sodium fluoride naf
Manufactured by Sangon
Sourced in China
Sodium fluoride (NaF) is a chemical compound that is commonly used as a laboratory reagent. It is a white, crystalline solid that is soluble in water. Sodium fluoride's primary function is to provide a source of fluoride ions for various chemical reactions and analyses.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using sodium fluoride naf
1
Spectrophotometric Assay for Enolase Inhibition
2
Protein Extraction and Western Blot Analysis of SGK1 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The tissues or cells were dissociated on ice and homogenized in radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) containing 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), 10 μL/mL sodium fluoride (NaF; Sangon Biotech, Shanghai, China), 2 µL/mL protease inhibitor cocktail (Merck-Millipore, Bedford, MA, USA), and 1 µL/mL sodium orthovanadate (Na2VO3; Sangon Biotech). The supernatant protein concentration was determined using the BCA method. Samples containing 30 µg of protein were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred onto a nitrocellulose filter membrane. The membrane was blocked with 5% fat-free milk for 1 hour at room temperature and incubated with rabbit anti-human SGK1 (1:1,000; Cell Signaling Technology, Danvers, MA, USA), and rabbit anti-human p-SGK1 (1:1,000; Cell Signaling Technology) overnight at 4°C. The membrane was then washed and incubated with a corresponding secondary antibody, and processed using an electrochemiluminescence reaction kit (Merck-Millipore), followed by exposure to medical film. The density of the target protein band relative to that of β-actin (1:10,000; Beyotime) was quantified using Bio-Rad Quantity One 1-D Analysis Software (Bio-Rad Laboratories, Hercules, CA, 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!