Knight:NuPAGE electrophoresis: Difference between revisions
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==Procedure== | ==Procedure== | ||
===Running buffer preparation=== | |||
Do this step first. | |||
#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 running the gel. | |||
#*Mix well. | |||
===Sample preparation=== | ===Sample preparation=== | ||
#Wells can accommodate 25μL loading volume. | #Wells can accommodate 25μL loading volume. | ||
Line 21: | Line 32: | ||
#*2 μL NuPAGE Reducing Agent | #*2 μL NuPAGE Reducing Agent | ||
#Heat samples at 70°C for 10 mins. | #Heat samples at 70°C for 10 mins. | ||
Set up the gel apparatus during the 10 mins sample heating step. | |||
Note that prestained molecular weight marker doesn't need any preparation. | |||
===Running the gel=== | ===Running the gel=== | ||
Line 39: | Line 44: | ||
#Gently pull the comb from the cassette in one smooth motion. | #Gently pull the comb from the cassette in one smooth motion. | ||
#Rinse the sample wells with 1X NuPAGE SDS running buffer. | #Rinse the sample wells with 1X NuPAGE SDS running buffer. | ||
#*Use a pipetman and pipet to squirt in running buffer. | |||
#Invert and shake to remove buffer. | #Invert and shake to remove buffer. | ||
#Repeat rinse two more times. | #Repeat rinse two more times. | ||
Line 49: | Line 55: | ||
#Load samples. | #Load samples. | ||
#Load protein molecular weight marker. | #Load protein molecular weight marker. | ||
#Fill lower buffer chamber with 600mL NuPAGE SDS running buffer. | #Fill lower buffer chamber at the gap near locking mechanism with 600mL NuPAGE SDS running buffer. | ||
#Run at 200V for | #Run at 200V for 30 minutes. | ||
===Staining the gel=== | |||
#Shut off the power. | |||
#Disconnect electrodes. | |||
#Remove gels. | |||
#Insert a knife in between the two plates and pry the plates apart. | |||
#*You should hear a cracking noise as you break the bond between the two plates. | |||
#Gently separate the two plates attempting to leave the gel on the bottom slotted plate. | |||
#Cut to separate gel from bottom lip. | |||
#Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below). | |||
You have two options for staining at this point. | |||
====Fast, microwave protocol==== | |||
#Add 100mL deionized water in staining tray. | |||
#Microwave staining tray loosely covered for 1 min on high. | |||
#Shake staining tray for 1 min on orbital shaker at room temperature. | |||
#Discard water. | |||
#Repeat steps 1-4 two more times. | |||
#Add 20mL SimplyBlue SafeStain. | |||
#Microwave staining tray loosely covered for 45 secs on high. | |||
#*1 min seemed a bit too long. | |||
#Shake staining tray for 5 mins on orbital shaker at room temperature. | |||
#Discard SimplyBlue SafeStain. | |||
#Add 100mL deionized water. | |||
#Shake staining tray for 10 mins on orbital shaker at room temperature. | |||
#Add 20mL 20% NaCl for at least 5 mins. | |||
#Gel can be stored in salt solution for several weeks. | |||
This protocol seems to work pretty well. | |||
====Slow, room temperature protocol==== | |||
#Add 100mL deionized water in staining tray. | |||
#Shake staining tray for 5 min on orbital shaker at room temperature. | |||
#Discard water. | |||
#Repeat steps 1-3 two more times. | |||
#Add 20mL SimplyBlue SafeStain. | |||
#Shake staining tray for 1 hr on orbital shaker at room temperature. | |||
#Add 100mL deionized water. | |||
#Shake staining tray for 1 hr or more on orbital shaker at room temperature. | |||
==References== | ==References== |
Revision as of 16:00, 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
Running buffer preparation
Do this step first.
- 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 running the gel.
- Mix well.
Sample preparation
- Wells can accommodate 25μL loading volume.
- Let sample buffer warm to room temperature.
- 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.
Set up the gel apparatus during the 10 mins sample heating step.
Note that prestained molecular weight marker doesn't need any preparation.
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.
- Use a pipetman and pipet to squirt in 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 at the gap near locking mechanism with 600mL NuPAGE SDS running buffer.
- Run at 200V for 30 minutes.
Staining the gel
- Shut off the power.
- Disconnect electrodes.
- Remove gels.
- Insert a knife in between the two plates and pry the plates apart.
- You should hear a cracking noise as you break the bond between the two plates.
- Gently separate the two plates attempting to leave the gel on the bottom slotted plate.
- Cut to separate gel from bottom lip.
- Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below).
You have two options for staining at this point.
Fast, microwave protocol
- Add 100mL deionized water in staining tray.
- Microwave staining tray loosely covered for 1 min on high.
- Shake staining tray for 1 min on orbital shaker at room temperature.
- Discard water.
- Repeat steps 1-4 two more times.
- Add 20mL SimplyBlue SafeStain.
- Microwave staining tray loosely covered for 45 secs on high.
- 1 min seemed a bit too long.
- Shake staining tray for 5 mins on orbital shaker at room temperature.
- Discard SimplyBlue SafeStain.
- Add 100mL deionized water.
- Shake staining tray for 10 mins on orbital shaker at room temperature.
- Add 20mL 20% NaCl for at least 5 mins.
- Gel can be stored in salt solution for several weeks.
This protocol seems to work pretty well.
Slow, room temperature protocol
- Add 100mL deionized water in staining tray.
- Shake staining tray for 5 min on orbital shaker at room temperature.
- Discard water.
- Repeat steps 1-3 two more times.
- Add 20mL SimplyBlue SafeStain.
- Shake staining tray for 1 hr on orbital shaker at room temperature.
- Add 100mL deionized water.
- Shake staining tray for 1 hr or more on orbital shaker at room temperature.