McClean:Western Blot

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:

1. Step 1
2. Step 2
3. Step 3

Protocol

1. Step 1
2. Step 2
3. Step 3

Notes

Please feel free to post comments, questions, or improvements to this protocol. Happy to have your input!

1. List troubleshooting tips here.
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3. 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.