McClean:Western Blot: Difference between revisions

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* Beta- mercaptoethanol
* Beta- mercaptoethanol
* 4X LDS sample buffer(Invitrogen NP0008)
* 4X LDS sample buffer(Invitrogen NP0008)
* Protease inhibitor(Roche catlog number: 11836170001)
* Protease inhibitor(Roche catalog number: 11836170001)
* Phosphatase inhibitor(Fisher catlog number: 78420)
* Phosphatase inhibitor(Fisher catalog number: 78420)
* 20X NuPAGE running buffer(Invitrogen NP0001)
* 20X NuPAGE running buffer(Invitrogen NP0001)
* Prestained Protein Ladder(Invitrogen catlog number:10748-010)
* Prestained Protein Ladder(Invitrogen catalog number:10748-010)
* Magic Marker(Invitrogen catlog number:LC5602)
* Magic Marker(Invitrogen catalog number:LC5602)
* NuPage pre-cast gel
* NuPage pre-cast gel
* PVDF membrane(Invitrogen catlog number:LC2005)
* PVDF membrane(Invitrogen catalog number:LC2005)
* Methanol
* Methanol
* 20X NuPAGE buffer(Invitrogen NP0006)
* 20X NuPAGE buffer(Invitrogen NP0006)
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* Nonfat dry milk
* Nonfat dry milk
* Primary and secondary antibody
* Primary and secondary antibody
* Pierce Supersignal Femto kit (Pierce # 34095)


==Stock Solutions==
==Stock Solutions==
Line 44: Line 45:
'''Running Gel and Transfer'''
'''Running Gel and Transfer'''
# 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.  
# 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 tighten them with the clamp.
# 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.
# Run gel at 200V until dye reaches the bottom of the gel.
# Run gel at 200V until dye reaches the bottom of the gel.
Line 52: Line 53:
# After gel is done running, crack open the gel and cut off the wells and gel bottom. Move gel to a container with 1X transfer buffer.
# After gel is done running, crack open the gel and cut off the wells and gel bottom. Move gel to a container with 1X transfer buffer.
# Assemble the blotting sandwich. Look at the picture in the Invitrogen manual. Be careful to remove bubbles trapped in the sandwich.
# Assemble the blotting sandwich. Look at the picture in the Invitrogen manual. Be careful to remove bubbles trapped in the sandwich.
# Put the sandwich into the tank and tighten with the clamp.
# Fill up the chamber with the 1X transfer buffer and outside with DI water.
# Run at cold room at 13V overnight.


'''Blotting'''
'''Blotting'''
Line 61: Line 66:
# Incubate on a rocker with secondary antibody solution at room temperature for 1 hour.
# Incubate on a rocker with secondary antibody solution at room temperature for 1 hour.
# Wash the membrane with TBST 4X5 minutes on a rocker.
# Wash the membrane with TBST 4X5 minutes on a rocker.
# Put the sandwich to the tank and tight with the clamp.
 
# Fill up the chamber with the 1X transfer buffer and outside with DI water.
'''Develop'''
# Run at cold room at 13V overnight.
# Mix two substrates 1:1 from Pierce Supersignal Femto kit (Pierce # 34095). (For one membrane use 1.5ml of each.)
# When finished washing, hold the corner of the membrane with forceps and allow TBST to drip off and put the membrane on the saran wrap with the protein side up.
# Spread the mixture onto the membrane on the protein side and incubate 5 minutes.
# Use forceps to lift the corner of the membrane and allow the solution to drip off. Try to drip off as much as you can here.
# Put the membrane into the sheet protector and use a roller roll on it to remove bubbles.
# Put the sheet protector into the film cassette and go to dark room to develop film.


==Notes==
==Notes==

Revision as of 10:04, 5 June 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 catalog number: 11836170001)
  • Phosphatase inhibitor(Fisher catalog number: 78420)
  • 20X NuPAGE running buffer(Invitrogen NP0001)
  • Prestained Protein Ladder(Invitrogen catalog number:10748-010)
  • Magic Marker(Invitrogen catalog number:LC5602)
  • NuPage pre-cast gel
  • PVDF membrane(Invitrogen catalog 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
  • Pierce Supersignal Femto kit (Pierce # 34095)

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 ( 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 tighten 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.
  6. Cover the membrane with 100% methanol and then pour the methanol back to a tube, add 1X transfer buffer to cover the membrane. Keep it in 4 degree.
  7. In a container, wet the transfer pads with 1X transfer buffer and keep them in 4 degree until ready to transfer.
  8. After gel is done running, crack open the gel and cut off the wells and gel bottom. Move gel to a container with 1X transfer buffer.
  9. Assemble the blotting sandwich. Look at the picture in the Invitrogen manual. Be careful to remove bubbles trapped in the sandwich.
  10. Put the sandwich into the tank and tighten with the clamp.
  11. Fill up the chamber with the 1X transfer buffer and outside with DI water.
  12. Run at cold room at 13V overnight.


Blotting

  1. Make 5% milk with dry nonfat milk and TBST(1L: 10ml of 1M Tris,pH8.0(Final concentration 10mM) , 60ml 0f 2.5M NaCl(Final concentration 150mM), 1ml Tween 20(Final concentration 0.1%) and add water up to 1L.)
  2. Rinse the membrane with TBST and then blot membrane in 5% milk on a rocker for 1 hour.
  3. Wash the membrane with TBST 4X5 minutes on a rocker.
  4. Incubate on a rocker with primary antibody solution at room temperature for 1 hour.
  5. Wash the membrane with TBST 4X5 minutes on a rocker.
  6. Incubate on a rocker with secondary antibody solution at room temperature for 1 hour.
  7. Wash the membrane with TBST 4X5 minutes on a rocker.

Develop

  1. Mix two substrates 1:1 from Pierce Supersignal Femto kit (Pierce # 34095). (For one membrane use 1.5ml of each.)
  2. When finished washing, hold the corner of the membrane with forceps and allow TBST to drip off and put the membrane on the saran wrap with the protein side up.
  3. Spread the mixture onto the membrane on the protein side and incubate 5 minutes.
  4. Use forceps to lift the corner of the membrane and allow the solution to drip off. Try to drip off as much as you can here.
  5. Put the membrane into the sheet protector and use a roller roll on it to remove bubbles.
  6. Put the sheet protector into the film cassette and go to dark room to develop film.

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.
  3. 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

or instead, discuss this protocol.


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