The SNARE proteins were essentially isolated as previously described (Mima et al., 2008 (link)). Vti1p and Nyv1p were gel-filtered into RB150/ß-OG (20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol [vol/vol], 1% [wt/vol] ß-octyl glucoside) after purification using Sephacryl S-200 HR (GE Healthcare Biosciences, Pittsburgh, PA). A complete detergent exchange was confirmed by determining the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations in elution fractions (Urbani and Warne, 2005 (link)); only fractions with no residual amounts of CHAPS were pooled and used in the reconstitution experiments. Ypt7p (Hickey et al., 2009 (link)), Sec17p (Schwartz and Merz, 2009 (link)), His6-Sec18p (Haas and Wickner, 1996 (link)), and HOPS (Hickey and Wickner, 2010 (link)) were isolated as previously described.
Protocol detail
Reconstitution of SNARE-Mediated Membrane Fusion
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 : Dartmouth College
Top 5 similar protocols
Protocol cited in 13 other protocols
Variable analysis
independent variables
- None explicitly mentioned
- None explicitly mentioned
- Vti1p and Nyv1p were gel-filtered into RB150/ß-OG (20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol [vol/vol], 1% [wt/vol] ß-octyl glucoside) after purification
- A complete detergent exchange was confirmed by determining the 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) concentrations in elution fractions; only fractions with no residual amounts of CHAPS were pooled and used in the reconstitution experiments
- Ypt7p, Sec17p, His6-Sec18p, and HOPS were isolated as previously described
- None specified
- None specified
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!