McClean:Western Blot: Difference between revisions

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==Protocol==
==Protocol==
'''Cell growth and protein extraction'''
'''Cell growth and protein extraction'''
# 1.Grow cells to mid-log (~1x107 cells/ml; A600 = 0.7 or klett 80) and collect 1.5 ml cells in 1.5 ml microfuge tube (1 minute, 14000xg). It is important not to grow the cells to a high density as this method will not work well.
# Grow cells to mid-log (~1x107 cells/ml; A600 = 0.7 or klett 80) and collect 1.5 ml cells in 1.5 ml microfuge tube (1 minute, 14000xg). It is important not to grow the cells to a high density as this method will not work well.
# 2.Resuspend cells in 100 microliters sample buffer(Bring 4X sample buffer to 1X with BME(final concertration 10%), protease inhibitor, phosphatase inhibitor and water).
# Resuspend cells in 100 microliters sample buffer(Bring 4X sample buffer to 1X with BME(final concertration 10%), protease inhibitor, phosphatase inhibitor and water).
# 3.Heat at 95 deg C for 5 minutes.
# Heat at 95 deg C for 5 minutes.
# 4.Vortex for 30 seconds.
# Vortex for 30 seconds.
# 5.Centrifuge 14000xg for 5 minutes.
# Centrifuge 14000xg for 5 minutes.


'''Running Gel and blotting'''
'''Running Gel and Transfer'''
# 1.Take Nupage pre-cast gel out, remove the white strip and comb, rinse with water.  
# Take Nupage pre-cast gel out, remove the white strip and comb, rinse with water.  
# 2.With the wells facing inward, place two gels or one gel and a buffer dam against the rubber gasket of the Nupage buffer core, put them into the tank(only fit one way) and tight them with the clamp.
# With the wells facing inward, place two gels or one gel and a buffer dam against the rubber gasket of the Nupage buffer core, put them into the tank(only fit one way) and tight them with the clamp.
# 3.Fill the chamber up with 1X Nupage running buffer and fill the outside with 1X Nupage running buffer to the level that the wire is covered.
# Fill the chamber up with 1X Nupage running buffer and fill the outside with 1X Nupage running buffer to the level that the wire is covered.
# 4.Run gel at 200V until dye reaches the bottom of the gel.
# Run gel at 200V until dye reaches the bottom of the gel.
# 5.In the meantime, prepare the transfer buffer. Bring up to 20% methanol, 1X transfer buffer in water. 500ml transfer buffer: 20X Transfer buffer 25ml, Methanol 100ml and Water 375ml. Keep it in 4 degree.
# In the meantime, prepare the transfer buffer. Bring up to 20% methanol, 1X transfer buffer in water. 500ml transfer buffer: 20X Transfer buffer 25ml, Methanol 100ml and Water 375ml. Keep it in 4 degree.


==Notes==
==Notes==

Revision as of 11:01, 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

  • 4X LDS sample buffer(Invitrogen NP0008)

Stock Solution 2

  • 20X NuPAGE running buffer(Invitrogen NP0001)

Stock Solution 3

  • 1M Tris, pH 8.0

Stock Solution 4

  • 2.5M NaCl

Protocol

Cell growth and protein extraction

  1. Grow cells to mid-log (~1x107 cells/ml; A600 = 0.7 or klett 80) and collect 1.5 ml cells in 1.5 ml microfuge tube (1 minute, 14000xg). It is important not to grow the cells to a high density as this method will not work well.
  2. Resuspend cells in 100 microliters sample buffer(Bring 4X sample buffer to 1X with BME(final concertration 10%), protease inhibitor, phosphatase inhibitor and water).
  3. Heat at 95 deg C for 5 minutes.
  4. Vortex for 30 seconds.
  5. Centrifuge 14000xg for 5 minutes.

Running Gel and Transfer

  1. Take Nupage pre-cast gel out, remove the white strip and comb, rinse with water.
  2. With the wells facing inward, place two gels or one gel and a buffer dam against the rubber gasket of the Nupage buffer core, put them into the tank(only fit one way) and tight them with the clamp.
  3. Fill the chamber up with 1X Nupage running buffer and fill the outside with 1X Nupage running buffer to the level that the wire is covered.
  4. Run gel at 200V until dye reaches the bottom of the gel.
  5. In the meantime, prepare the transfer buffer. Bring up to 20% methanol, 1X transfer buffer in water. 500ml transfer buffer: 20X Transfer buffer 25ml, Methanol 100ml and Water 375ml. Keep it in 4 degree.

Notes

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

  1. List troubleshooting tips here.
  2. You can also link to FAQs/tips provided by other sources such as the manufacturer or other websites.
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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

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