NuPAGE Gel Electrophoresis 蛋白电泳[Kitto Lab, The University of Texas at Austin

A gel electrophoresis system used for SDS-PAGE protein analysis. The gels are made up of Bis-Tris-HCl (pH 6.4) polyacrylamide and are intended for denaturing conditions only.

NuPAGE Electrophoresis Protocols

1) Remove gel from pouch and rinse gel with D.I. water holding the gel by the edges of the cassette. (Gels should be stored at 4º C)

2) Mark the bottom of the lanes with a permanent marker.

3) Peel off tape from the bottom of the cassette.

4) Insert gel into the Mini-Cell so that you can read "10 % BIS-TRIS" when facing the gel. If only running one gel use the square plastic dam to replace the second gel cassette.

5) Check for tightness of the seal by filling the inner chamber with a small amount of buffer. If a leak is detected reposition or reassemble the chamber.

6) Remove the comb from the gel. Using a disposable needle tip, remove any residual buffer from the wells.

7) Proceed to fill the inner chamber with running buffer so that the buffer level covers the level of the wells. If running reduced samples add .5 mL of NuPAGE Anti-oxidant.

8) Load samples onto gel and pour running buffer into the outer chamber until it covers about half of the cassette.

9) Run gels according to the protocol which is applicable:

10% Bis-Tris Gel with MES Running Buffer

Voltage: 200V constant

Expected Current: Start: 110-125 mA per gel; End: 70-80 mA per gel

Approx. Run Time: 35 minutes

10% Bis-Tris Gel with MOPS Running Buffer

Voltage: 200 V constant

Expected Current: Start: 110-115 mA per gel; End: 60-70 mA per gel

Approx. Run Time: 50 minutes

4-12% Bis-Tris Gel with MES Running Buffer

Voltage: 200V constant

Expected Current: Start: 110-125 mA per gel; End: 70-80 mA per gel

Approx. Run Time: 35 minutes

4-12% Bis-Tris Gel with MOPS Running Buffer

Voltage: 200V constant

Expected Current: Start: 110-115 mA per gel; End 70-80 mA per gel

Approx. Run Time: 50 minutes

10) When gel is complete, shut off power, disconnect electrodes and remove gel from Mini-Cell

11) Separate each of the three bonded sides by prying them open with a screw driver. Using a razor blade cut the sides of the gel. The thick edge at the bottom of the gel should be cut off.

Sample Preparation

1) Prepare sample buffer by mixing 50 µL of NuPAGE Sample Buffer, 30 µL D.I. water, and 20µL of reducing agent. (Reducing agent can be either 0.5 M DTT in stabilized liquid form or beta-mercaptoethanol)

2) Add sample buffer in a 1:1 ratio to sample

3) Heat mixture of sample buffer and sample for 5 minutes in boiling water bath.

4) Heat See-Blue standard for 3 minutes in boiling water bath.