McClean:Western Blot
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Overview
This is a protocol to perform rapid yeast protein prep for SDS PAGE and Western.
Materials
- Beta- mercaptoethanol
- 4X LDS sample buffer(Invitrogen NP0008)
- Protease inhibitor(Roche catlog number: 11836170001)
- Phosphatase inhibitor(Fisher catlog number: 78420)
- 20X NuPAGE running buffer(Invitrogen NP0001)
- Prestained Protein Ladder(Invitrogen catlog number:10748-010)
- Magic Marker(Invitrogen catlog number:LC5602)
- NuPage pre-cast gel
- PVDF membrane(Invitrogen catlog number:LC2005)
- Methanol
- 20X NuPAGE buffer(Invitrogen NP0006)
- 1M Tris, pH 8.0
- 2.5M NaCl
- Tween 20
- Nonfat dry milk
- Primary and secondary antibody
Stock Solutions
Stock Solution 1
- This is a very simple solution, so we only need a one line description of how to make it.
Stock Solution 2
This is a more involved solution, so we will describe how to make it in several steps:
- Step 1
- Step 2
- Step 3
Protocol
- Step 1
- Step 2
- Step 3
Notes
Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!
- List troubleshooting tips here.
- You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
- Anecdotal observations that might be of use to others can also be posted here.
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References
Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96.
Contact
- Megan N McClean 14:01, 20 July 2011 (EDT)
or instead, discuss this protocol.