McClean:Western Blot: Difference between revisions
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==Materials== | ==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 Solutions== |
Revision as of 09:20, 30 May 2013
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.
Please sign your name to your note by adding '''*~~~~''': to the beginning of your tip.
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.