Cells were homogenized in RIPA lysis buffer (20 mM Tris, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% sodium deoxycholate, 1% Triton X-100 (TX-100), and 0.1% SDS, pH 7.4), containing a protease inhibitor cocktail (1:100, Sigma) and phosphatase inhibitor cocktails 1 and 3 (both at 1:200, Sigma). Lysates were cleared by centrifugation (20 min at 14,000×g), diluted with 2× SDS sample buffer and boiled. SDS-PAGE and immunoblotting were conducted by standard protocols with an equal amount of total protein (10 or 20 μg) per lane. Results shown are representative immunoblots of at least three independent experiments. Protein expression was quantified by densitometry, and signal intensities were calculated using ImageJ software. The densitometry data are presented as normalized values, with the expression level of control groups set at 1 arbitrary unit.
Phosphatase inhibitor cocktails 1 and 3
Manufactured by Merck Group
Phosphatase inhibitor cocktails 1 and 3 are laboratory reagents used to inhibit the activity of phosphatases, which are enzymes that remove phosphate groups from proteins. These cocktails contain a combination of different phosphatase inhibitors to provide a broad spectrum of inhibition. They are commonly used in biological research to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
3 protocols using phosphatase inhibitor cocktails 1 and 3
1
Cell Lysis and Protein Analysis
2
Whole Cell Lysate Preparation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell lysates were prepared by removing the media, washing the cells with PBS, scraping the cells free with a cell lifter and then pelleting them by centrifugation at 3000 rpm for 10 minutes. Cell pellets were resuspended in lysis buffer containing 20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, protease inhibitor cocktail (Sigma, St. Louis, MO), and phosphatase inhibitor cocktails 1 and 3 (Sigma, St. Louis, MO). Lysates were clarified and stored at −80°C. 30 µg of protein was immunoblotted per our previously described protocol [27] (link). All primary antibodies were diluted to a final concentration of 1∶1000 except GAPDH and LAMIN (1∶5000). Immunoreactive species were detected by enhanced chemiluminescence (Pierce ECL; Thermo Scientific, Waltham, MA). Nuclear and cytoplasmic fractions were extracted using the NE-PER extraction kit (Thermo Scientific, Waltham, MA) following the manufacturers' instructions.
3
Western Blot Analysis of Cellular Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blot analyses, protein was harvested from cells plated to 70-80% confluence. Cells were homogenized in RIPA lysis buffer (20 mM tris, 50 mM NaCl, 2 mM EDTA, 2 mM EGTA, 1% sodium deoxycholate, 1% Triton X-100 (TX-100), and 0.1% SDS, pH 7.4), containing a protease inhibitor cocktail (1:100, Sigma) and phosphatase inhibitor cocktails 1 and 3 (both at 1:200, Sigma). Lysates were cleared by centrifugation (20 min at 14,000g), diluted with 2 Â SDS sample buffer and boiled.
Immunoblotting was conducted by standard protocols with an equal amount of total protein (10 mg) per lane. The protein extracts were loaded, size-fractionated by SDS-polyacrylamide gel electrophoeresis and transferred to PVDF membranes (Bio-Rad Laboratories). After blocking, the membranes were incubated with the specific rabbit polyclonal antibodies in dilution buffer at 4°C overnight. The blotted membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:1,000) at room temperature for 2 hr. Subsequently, the targeting protein expression level was detected and visualized using the Enhanced Chemiluminescence (ECL) detection system. Primary antibodies Cyclin A1 (H-230) and CEP55 (H-300) (Santa Cruz Biotechnology) were used at 1:500 dilution. GAPDH was used as the internal control.
Immunoblotting was conducted by standard protocols with an equal amount of total protein (10 mg) per lane. The protein extracts were loaded, size-fractionated by SDS-polyacrylamide gel electrophoeresis and transferred to PVDF membranes (Bio-Rad Laboratories). After blocking, the membranes were incubated with the specific rabbit polyclonal antibodies in dilution buffer at 4°C overnight. The blotted membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:1,000) at room temperature for 2 hr. Subsequently, the targeting protein expression level was detected and visualized using the Enhanced Chemiluminescence (ECL) detection system. Primary antibodies Cyclin A1 (H-230) and CEP55 (H-300) (Santa Cruz Biotechnology) were used at 1:500 dilution. GAPDH was used as the internal control.
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!