50 mL cultures from each strain were harvested by centrifugation at 8000 rpm, 4°C for 10 min. The pellets were then washed with 10 mL of 0.9% NaCl. Cells were suspended in 2 mL 50 mM Tris-HCl, 10 mM CaCl2, and 0.1% Nonidet P-40 ( ) supplemented with 20 μL protease inhibitor cocktail (Sigma, USA). Cells were lysed by tip-probe sonication through 20 cycles of sonication with 15 s on and 60 s off on ice. The lysates were centrifuged at 12000 rpm for 10 min at 4°C. Supernatants were collected and stored at -80°C for future proteomics. The protein concentration was measured through Pierce Bradford Protein Assay (Thermo Fisher) with standards following manufacturer’s instructions. Samples from each strain were prepared from three biological replicates.
Pierce bradford protein assay
Manufactured by Thermo Fisher Scientific
The Pierce Bradford Protein Assay is a colorimetric assay used to determine the concentration of proteins in a sample. It is based on the Bradford dye-binding method, which utilizes the binding of Coomassie Brilliant Blue G-250 dye to proteins. The change in absorbance of the dye-protein complex is measured at 595 nm, and the protein concentration is determined by comparison to a standard curve.
Automatically generated - may contain errors
2 protocols using pierce bradford protein assay
1
Proteome Extraction from Bacterial Strains
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed twice with 1X PBS and harvested with RIPA lysis buffer (Sigma Cat # R0278) containing protease inhibitors (Sigma Cat # P8340) and phosphatase inhibitors (Roche Cat # 4906845001). Whole cell lysates were centrifuged at 10,000 rpm for 10 minutes at 4°C. Supernatants were transferred to fresh tubes and protein concentration was quantified using the Pierce Bradford protein assay (Thermo Fisher Cat # 23225). Thirty μg of protein was analyzed on a 10% SDS-polyacrylamide gel by SDS-PAGE electrophoresis and transferred to a PVDF membrane (Biorad Cat #1620177). Membranes were blocked in 1X TBS buffer containing 5% BSA and 0.001% Tween for 1 hour at room temperature, and then incubated with primary antibody Phospho-SAPK/JNK (1:1000), SAPK/JNK (1:1000), Phospho-c-Jun (1:1000), C-Jun (1:1000), Phospho-Erk1/2 (1:1000), Erk1/2 (1:1000), PSA (1:3000), AR (1:1000), or GAPDH (1:5000) overnight at 4°C. Blots were washed with TBST three times for 5 minutes each and incubated with secondary antibodies (anti-rabbit IgG – Abcam # ab6721 1:10,000) for 1 hour at room temperature. Signals were visualized using SuperSignal West Pic PLUS chemiluminescence substrate (Pierce Cat #34580) according to the manufacturer's instructions (Thermo Fisher Scientific) and captured on film. Quantitative measurements of Western blot analysis were performed using Fiji and GraphPad software (Prism 7).
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!