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MOLECULAR BIOLOGY: WORKING WITH PROTEINS

PROTEIN DETECTION: GEL ELECTROPHORESIS

RUNNING NOVEX NuPage GELS

Running Novex NuPage Gels
Contributor: The Laboratory of Steve Hahn at the Fred Hutchinson Cancer Research Center
 
Overview
These are simplified instructions for use of Novex precast NuPage gels. The XCEll II™ mini cell apparatus is required for this protocol. For more information on Novex gels and products see their webpage http://www.invitrogen.com/catalog_novex.html
 
Procedure
A. Preparation for SDS-PAGE

1. Select the desired Running Buffer, either MOPS/SDS Running Buffer for proteins greater than 200 kDa to 14 kDa or MES/SDS Running Buffer for proteins between 25 to 60 kDa.

2. Prepare 800 ml of the Running Buffer (1X).

3. Remove the precast gel from the bag. Rinse the gel with ddH2O.

4. Peel off the tape on the back of the gel and remove the comb carefully.

5. Wash out the wells a total of three times with 1X Running Buffer using a pasteur pipette. Fill the sample wells with buffer and remove all air bubbles.

6. Set up the XCEll II™ mini-cell apparatus as per manufacturer's instructions.

7. Fill the upper buffer chamber with about 200 ml of Running Buffer and check for leaks. If there are no leaks, add the remainder of the Running Buffer to the lower buffer chamber.

8. For reducing SDS-PAGE, add 0.5 ml of antioxidant (ie DTT or 2-Mercaptoethanol) to the 200 ml of Running Buffer in the upper chamber. For optimum results add within 15 min of actually running the gel. This helps keep the samples reduced as the gel runs (see Hint #2).

9. For MOPS buffer, run at a constant 200Volts for approximately 50 min. Expect 100 to 115 mA current at the beginning and 60 to 70 mA at the end of the run.

10. For MES buffer, run at a constant 200 Volts for approximately 35 min. Expect 110 to 125 mA current at the beginning and 70 to 80 mA at the end of the run.

B. SDS PAGE Sample Preparation

1. To prepare the protein sample, add 1X NuPAGE Sample Buffer (see Hint #3).

2. Add one-tenth volume of 0.5 M DTT.

3. Heat the samples for 10 min at 70°C (see Hint #4).

4. Load the samples on the NuPAGE gel and carry out the electrophoresis as outlined above in Section A (see Hint #5).

C. Electroblotting

1. Prepare 400 ml of 1X Working NuPAGE Transfer Buffer (see Hint #7).

2. Carefully prewet a 0.2 μm PVDF membrane for 30 sec in Methanol.

3. Rinse the wet membrane briefly in ddH2O.

4. Soak the membrane in approximately 50 ml of 1X Working NuPAGE Transfer Buffer for 5 to 10 min (see Hint #8).

5. Presoak the blotting pads and two pieces of the manufacturer recommended filter paper in 1X Working NuPAGE Transfer Buffer. Ensure that there are no air bubbles.

6. After the electrophoresis is complete, crack open the gel cassette with a blunt edged knife or spatula to expose the polyacrylamide gel.

7. Remove the stacking gel (containing the wells) with a scalpel.

8. Place a piece of presoaked filter paper on the gel, leaving the lower edge of the gel uncovered. Ensure that there are no trapped air bubbles.

9. Turn the cassette over onto a piece of parafilm or plastic wrap. Cut the foot off the gel using a scalpel and transfer the gel onto the parafilm or plastic wrap.

10. Wet the surface of the gel with 1X Working NuPAGE Transfer Buffer and position the presoaked PVDF membrane on the gel. Use a clean pipette to roll out any air bubbles trapped between the membrane and the gel.

11. Place a piece of presoaked filter paper on top of the PVDF membrane.

12. Assemble the sandwich in the transfer apparatus according to the manufacturer's instructions. Fill the chamber with 1X Working NuPAGE Transfer Buffer until the gel/membrane is just covered.

13. Fill the outer buffer chamber with 650 ml ddH2O to dissipate the heat during the transfer.

14. Run the unit at a constant 24 Volts for between 90 min to 2 hr. Expect approximately 130 mA current at the start and 115 mA current at the end of the transfer (see Hint #9).

Solutions
NuPAGE Sample Buffer (4X)   0.66 g Tris-HCl
0.0075 g Serva Blue
0.68 g Tris Base
0.006 g EDTA
0.0025 g Phenol Red
0.8 g SDS
The 1X Buffer should be pH 7.2 (Do not adjust with Acid or Base)
4.0 g Sucrose
Add ddH2O to 10 ml
1X Working NuPAGE Transfer Buffer   Add ddH2O to a final volume of 400 ml (See Hint #6)
48 to 60 ml of Methanol (to a final concentration of 12% to 15% (v/v) final)
20 ml of NuPAGE Transfer Buffer (20X)
CAUTION! see Hint #1
NuPAGE Transfer Buffer (20X)   500 mM Bis-Tris
Store at 4°C
20.5 mM EDTA
500 mM Bicine
The 1X Buffer should be pH 7.2 (Do not adjust with Acid or Base)
MES/SDS Running Buffer (20X)   1 M Tris Base
1 M MES
The 1X Buffer should be pH 7.3 (Do not adjust with Acid or Base)
20.5 mM EDTA
69.3 mM SDS
MOPS/SDS Running Buffer (20X)   1 M Tris Base
The 1X Buffer should be pH 7.7 (Do not adjust with Acid or Base)
1 M MOPS
20.5 mM EDTA
69.3 mM SDS
100% Methanol
0.5 M DTT
 
BioReagents and Chemicals
Serva Blue
Tris-Cl
SDS
Sucrose
MES
EDTA
Tris Base
DTT
Phenol Red
Methanol
Bis-Tris
Bicine
MOPS
 
Protocol Hints
1. CAUTION! This substance is a biohazard. Consult this agent's MSDS for proper handling instructions.

2. The DTT in the sample does not migrate in the neutral pH gels as it does in Tris-Glycine gels.

3. This is very viscous and needs to be heated to at least room temperature.

4. Boiling for 2 to 3 min works as well.

5. Prestained protein standards migrate abnormally in NuPAGE gels. Use See-Blue pre stained marker proteins and use the NOVEX callibration chart to determine molecular weight of your sample. The gels are 1.0 mm thick. A 15 well comb can hold 15 μl per well and the 12 well comb can hold 20 μl in each well

6. Increased Methanol concentration will aid in protein binding to the membrane, but may inhibit the

protein elution from the gel. In general, use the lower Methanol concentration if your protein is large or has a problem transferring out of the gel.

7. The contributors of this protocol suggest not transferring two gels simultaneously in the same apparatus.

8. Do not presoak the gel.

9. For large proteins which have a problem eluting from the gel, the transfer can be run for as much as 2.5 hr. Be aware that longer transfer times may cause smaller proteins to pass through the membrane.