McClean:Western Blot: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
Line 19: | Line 19: | ||
* Nonfat dry milk | * Nonfat dry milk | ||
* Primary and secondary antibody | * Primary and secondary antibody | ||
* Pierce Supersignal Femto kit (Pierce # 34095) | |||
==Stock Solutions== | ==Stock Solutions== |
Revision as of 12:53, 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
- 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
- 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.
- Resuspend cells in 100 microliters sample buffer(Bring 4X sample buffer to 1X with BME(final concertration 10%), protease inhibitor, phosphatase inhibitor and water).
- Heat at 95 deg C for 5 minutes.
- Vortex for 30 seconds.
- Centrifuge 14000xg for 5 minutes.
Running Gel and Transfer
- 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 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.
- Run gel at 200V until dye reaches the bottom of the gel.
- 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.
- 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.
- In a container, wet the transfer pads with 1X transfer buffer and keep them in 4 degree until ready to transfer.
- 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.
- 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
- 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.)
- Rinse the membrane with TBST and then blot membrane in 5% milk on a rocker for 1 hour.
- Wash the membrane with TBST 4X5 minutes on a rocker.
- Incubate on a rocker with primary antibody solution at room temperature for 1 hour.
- Wash the membrane with TBST 4X5 minutes on a rocker.
- Incubate on a rocker with secondary antibody solution at room temperature for 1 hour.
- Wash the membrane with TBST 4X5 minutes on a rocker.
Develop
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.