Comprehensive Western Blotting Procedure
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization :
Other organizations : Yonsei University
Protocol cited in 1 other protocol
Variable analysis
- Slight modifications to the Western blotting protocol described in Xie et al., 2017
- Protein expression levels of HA, FLAG, MYC, BCL-xL, MCL-1, ACTIN, GAPDH, BCL2, BAX, and PARP1
- Cell lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 1mM EDTA pH 8.0, and protease cocktail inhibitor)
- Centrifugation (14,000 × g for 20 min at 4°C)
- SDS-PAGE (6–15%)
- Nitrocellulose membranes (GE Healthcare)
- Blocking buffer (3% dried skim milk in TBST: 50 mM Tris pH 8.0, 150 mM NaCl, and 0.5% Tween 20)
- Primary antibodies: anti-HA, anti-FLAG, anti-MYC, anti-BCL-xL, anti-MCL-1, anti-ACTIN, anti-GAPDH, anti-BCL2, anti-BAX, and anti-PARP1
- Peroxidase-conjugated secondary antibodies
- Enhanced chemiluminescence (ECL) detection
- Positive controls: Not explicitly mentioned
- Negative controls: Not explicitly mentioned
Annotations
Based on most similar protocols
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!