From 2007.igem.org

Revision as of 12:25, 29 September 2007 (view source) Craig (Talk | contribs) (→Primary & secondary Reagents Required including controls) ← Older edit		Latest revision as of 12:35, 29 September 2007 (view source) Craig (Talk | contribs)
(One intermediate revision not shown)
Line 5:		Line 5:
	*Time to complete protocol:		*Time to complete protocol:
-	**Lab time: 15min.	+	**Lab time: 15min, 5min, 5min
-	**Waiting time:2h	+	**Waiting time:2h, 1h, 30min
	*Approximate cost of materials: $		*Approximate cost of materials: $
Line 12:		Line 12:
	=====Primary & secondary Reagents Required including controls=====		=====Primary & secondary Reagents Required including controls=====
	*[[Melbourne/Preparing a poly-acrylamide gel|polyacrylamide gel]]		*[[Melbourne/Preparing a poly-acrylamide gel|polyacrylamide gel]]
-	*1X SDS buffer	+	*1X SDS buffer (6.05g [[Melbourne/primary Tris|Tris]], 28.83g glycine, 2g SDS per liter)
	*Benchmark Protein Ladder		*Benchmark Protein Ladder
	*Denatured protein samples		*Denatured protein samples
-	*Commassie blue	+	*Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie)
	*Destain solution (10% methanol, 10% acetic acid)		*Destain solution (10% methanol, 10% acetic acid)

---

Latest revision as of 12:35, 29 September 2007

<Return to list of protocols> <Team home page>

• Applications:
• Separation of proteins on a gel
• Western blots

• Time to complete protocol:
  • Lab time: 15min, 5min, 5min
  • Waiting time:2h, 1h, 30min
• Approximate cost of materials: $

Contents 1 Method from primary and secondary reagents 1.1 Primary & secondary Reagents Required including controls 1.2 Method including controls 1.3 Equipement Required 1.4 References

Method from primary and secondary reagents

Primary & secondary Reagents Required including controls

• polyacrylamide gel
• 1X SDS buffer (6.05g Tris, 28.83g glycine, 2g SDS per liter)
• Benchmark Protein Ladder
• Denatured protein samples
• Commassie blue (50% methanol, 10% acetic acid, 0.2% commassie)
• Destain solution (10% methanol, 10% acetic acid)

Method including controls

1. Take the comb out of the prepared polyacrylamide gel. Place the gel in a holder and attach the holder to the PAGE apparatus.
2. Insert the apparatus in the tank and pour 1X SDS buffer in the tank and inside the apparatus.
3. Pipette 10ul of Benchmark ladder in one lane of the gel
4. Pipette desired amounts of protein samples in other lanes of the gel.
5. Put the lid on and connect the cables to the power supply
6. Set the power supply to 80V. Run for 20 minutes or until the dye front passes the stack.
7. Increase the voltage to 120V and continue running until the dye front reaches the end of the gel.
8. Cut off the power supply and remove gel from the apparatus.
9. Stain gel in commasie blue stain for 1h
10. Destain in destain solution for 30min.

Equipement Required

• Pipette and tips
• SDS PAGE apparatus
• Power source

References