Cells were lysed using a modified RIPA buffer (50 mM Tris (pH 8.0), 150 mM NaCl, 1.0% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 10mM Na4P2O7, 10 mM β-glycerolphosphate, 10mM NaF, 10mM EDTA, 1mM EGTA) with 1X HALT phosphatase/protease inhibitor cocktail (Thermo) added prior to lysis as described [35 (link)]. The following antibodies were used: Sigma, anti-Cx43 (C6219); Cell Signaling Technology, total ERK (9102L), phospho-ERK (9101L), total PKCδ (2058S), phospho-PKCδ Thr505 (9374S), PCNA (2586S), anti-Rabbit HRP (7074S), anti mouse HRP (7076S); Millipore, GAPDH (MAB374) and anti-osterix (AB3743). Representative blots are shown and were repeated a minimum of two times.
Protocol detail
Protein Extraction and Western Blotting
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.
Antibodies
Buffer
Cells
Cx43
Edta
Egta
Gapdh
Glycerolphosphate
Mouse
Na4p2o7
Nacl
Np 40
Pcna
Phosphatase
Protease inhibitor
Rabbit
Ripa
Sodium deoxycholate
Tris
Corresponding Organization : University of Maryland, Baltimore
Other organizations : Johns Hopkins University
Top 5 similar protocols
Variable analysis
independent variables
- Lysis buffer composition (50 mM Tris (pH 8.0), 150 mM NaCl, 1.0% NP-40, 0.5% Sodium deoxycholate, 0.1% SDS, 10mM Na4P2O7, 10 mM β-glycerolphosphate, 10mM NaF, 10mM EDTA, 1mM EGTA)
- Expression levels of Cx43, total ERK, phospho-ERK, total PKCδ, phospho-PKCδ Thr505, PCNA, GAPDH, osterix
- 1X HALT phosphatase/protease inhibitor cocktail (Thermo) added prior to lysis
- No positive or negative controls were explicitly mentioned.
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
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!