Knight:NuPAGE electrophoresis: Difference between revisions
From OpenWetWare
Jump to navigationJump to search
No edit summary |
No edit summary |
||
Line 30: | Line 30: | ||
#*Add 500μL NuPAGE Antioxidant immediately before use. | #*Add 500μL NuPAGE Antioxidant immediately before use. | ||
#*Mix well. | #*Mix well. | ||
===Running the gel=== | |||
#Wear gloves. | |||
#Remove the NuPAGE gel from the pouch. | |||
#Rinse the gel cassette with deionized water. | |||
#Peel the tape from the bottom of the cassette. | |||
#Gently pull the comb from the cassette in one smooth motion. | |||
#Rinse the sample wells with 1X NuPAGE SDS running buffer. | |||
#Invert and shake to remove buffer. | |||
#Repeat rinse two more times. | |||
#Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core. | |||
#Seat the gels on the bottom of the Mini-Cell and lock into place with the gel tension wedge. | |||
#*Use the plastic buffer dam if you are only running one gel. | |||
#Fill the upper buffer chamber with a small amount of uppder buffer chamber running buffer (with antioxidant) to check tightness of seal. | |||
#*If there is a leak, discard buffer, reseal chamber and try again. | |||
#Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL | |||
#Load samples. | |||
#Load protein molecular weight marker. | |||
#Fill lower buffer chamber with 600mL NuPAGE SDS running buffer. | |||
#Run at 200V for 35 minutes. | |||
==References== | ==References== |
Revision as of 11:09, 17 July 2006
In progress! For a better version of this protocol, see Sauer:bis-Tris SDS-PAGE, the very best.
Purpose
To run a denaturing protein gel. Note that a preferable version of this protocol is available at Sauer:bis-Tris SDS-PAGE, the very best (recommended by Kathleen). But since we seem to already have the Invitrogen kit contents in lab, this protocol describes use of the Invitrogen system.
Materials
- NuPAGE Novex 10% Bis-Tris Gel 1.0 mm, 10 well
- XCell SureLock Mini-Cell electrophoresis apparatus
- 20X MES Running Buffer
- NuPAGE LDS Sample Buffer (4X)
- NuPAGE Reducing Agent (10X)
- NuPAGE Antioxidant
Procedure
Sample preparation
Wells can accommodate 25μL loading volume.
- Each sample should contain
- 13 μL protein sample (max 0.5μg per band)
- 5 μL NuPAGE LDS Sample Buffer
- 2 μL NuPAGE Reducing Agent
- Heat samples at 70°C for 10 mins.
Running buffer preparation
- Prepare 1000mL 1X NuPAGE SDS running buffer
- 50mL 20X MES Running Buffer
- 950mL deionized water
- Mix well.
- Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
- Add 500μL NuPAGE Antioxidant immediately before use.
- Mix well.
Running the gel
- Wear gloves.
- Remove the NuPAGE gel from the pouch.
- Rinse the gel cassette with deionized water.
- Peel the tape from the bottom of the cassette.
- Gently pull the comb from the cassette in one smooth motion.
- Rinse the sample wells with 1X NuPAGE SDS running buffer.
- Invert and shake to remove buffer.
- Repeat rinse two more times.
- Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
- Seat the gels on the bottom of the Mini-Cell and lock into place with the gel tension wedge.
- Use the plastic buffer dam if you are only running one gel.
- Fill the upper buffer chamber with a small amount of uppder buffer chamber running buffer (with antioxidant) to check tightness of seal.
- If there is a leak, discard buffer, reseal chamber and try again.
- Fill upper buffer chamber. Buffer level should exceed level of the wells. Requires about 200mL
- Load samples.
- Load protein molecular weight marker.
- Fill lower buffer chamber with 600mL NuPAGE SDS running buffer.
- Run at 200V for 35 minutes.