Western blotting was performed as described previously with a modification (38 ). Briefly, protein extracts were prepared using lysis buffer (7 M urea, 2 M thiourea, 2% (w/v) DTT) in the presence of 1% (v/w) protease inhibitor mixture (Pierce Biotechnology). The proteins were separated by SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in a TBST solution for 2 h at room temperature and incubated overnight at 4°C with primary antibodies. The membranes were washed with TBST buffer three times and incubated at room temperature for 2 h with secondary antibodies. The signals of the detected proteins were visualized with the SuperSignal West Femto Chemiluminescent Substrate Western Blotting detection system (Thermo Scientific).
Supersignal west femto chemiluminescent substrate western blotting detection system
Manufactured by Thermo Fisher Scientific
The SuperSignal West Femto Chemiluminescent Substrate is a Western Blotting detection system that provides high sensitivity for the detection of target proteins. The product is designed to generate a luminescent signal upon reaction with the target protein.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using supersignal west femto chemiluminescent substrate western blotting detection system
1
Western Blot Protein Analysis Protocol
2
Western Blot Protocol with Quantitative Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was conducted using a slightly modified version of a previously published protocol (Zheng et al., 2015 (link)). Briefly, a lysis buffer (7M urea, 2M thiourea, 2% (w/v) DTT) containing a 1% (v/w) protease inhibitor mixture (Pierce Biotechnology) was used to extract proteins, which were subsequently separated via SDS-PAGE and transferred to PVDF membranes. These blots were then blocked for 2 h at room temperature with 5% nonfat milk in TBST, followed by overnight (>12 h) incubation with primary antibodies at 4 °C. Anti-CCT6B was used at a 1:1000 dilution, while anti-β-TUBULIN was used at a 1:3000 dilution. After three washes with TBST, blots were then probed for 2 h with secondary antibodies, after which the SuperSignalWest Femto Chemiluminescent Substrate Western Blotting detection system (Thermo Scientific) was used to detect protein bands.
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!