Knight:NuPAGE electrophoresis

For a much cheaper 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 (for small proteins e.g. 13kD) or NuPAGE Novex 4-12% Bis-Tris Gel 1.0 mm, 10 well (for medium proteins e.g. 50-60kD)
 * XCell SureLock Mini-Cell electrophoresis apparatus
 * 20X MES or MOPS Running Buffer
 * NuPAGE LDS Sample Buffer (4X)
 * NuPAGE Reducing Agent (10X)
 * NuPAGE Antioxidant
 * SeeBlue Plus2 Pre-Stained Standard
 * Invitrogen SimplyBlue SafeStain

Running buffer preparation
Do this step first.


 * 1) Prepare 1000mL 1X NuPAGE SDS running buffer
 * 2) *50mL 20X MES Running Buffer (for very small proteins e.g. 13kD) or 20X MOPS Running Buffer (for medium-sized proteins e.g. 50-60kD).
 * 3) *950mL deionized water
 * 4) Mix well.
 * 5) Set aside 200mL buffer for use in Upper (Inner) buffer chamber).
 * 6) *Add 500&mu;L NuPAGE Antioxidant immediately before running the gel.
 * 7) *Mix well.

Sample preparation

 * 1) Wells can accommodate 25&mu;L loading volume.
 * 2) *Likely they can accommodate more ... maybe 40&mu;L.
 * 3) Let sample buffer warm to room temperature.
 * 4) Each sample should contain
 * 5) *13 &mu;L protein sample (max 0.5&mu;g per band)
 * 6) *5 &mu;L NuPAGE LDS Sample Buffer
 * 7) *2 &mu;L NuPAGE Reducing Agent
 * 8) Heat samples at 70&deg;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

 * 1) Wear gloves.
 * 2) Remove the NuPAGE gel from the pouch.
 * 3) Rinse the gel cassette with deionized water.
 * 4) Peel the tape from the bottom of the cassette.
 * 5) Gently pull the comb from the cassette in one smooth motion.
 * 6) Rinse the sample wells with 1X NuPAGE SDS running buffer.
 * 7) *Use a pipetman and pipet to squirt in running buffer.
 * 8) Invert and shake to remove buffer.
 * 9) Repeat rinse two more times.
 * 10) Orient the two gels in the Mini-Cell such that the notched "well" side of the cassette faces inward towards the buffer core.
 * 11) Seat the gels on the bottom of the Mini-Cell and lock into place with the gel tension wedge.
 * 12) *Use the plastic buffer dam if you are only running one gel.
 * 13) Fill the upper buffer chamber with a small amount of upper buffer chamber running buffer (with antioxidant) to check tightness of seal.
 * 14) *If there is a leak, discard buffer, reseal chamber and try again.
 * 15) Fill upper buffer chamber. Buffer level should exceed level of the wells.  Requires about 200mL
 * 16) Load samples.
 * 17) Load protein molecular weight marker (20 &mu;L per lane but 10&mu;L also seems to work).
 * 18) Fill lower buffer chamber at the gap near locking mechanism with 600mL NuPAGE SDS running buffer.
 * 19) Run at 200V for 30 minutes.

Staining the gel

 * 1) Shut off the power.
 * 2) Disconnect electrodes.
 * 3) Remove gels.
 * 4) Insert a knife in between the two plates and pry the plates apart.
 * 5) *You should hear a cracking noise as you break the bond between the two plates.
 * 6) Gently separate the two plates attempting to leave the gel on the bottom slotted plate.
 * 7) Cut to separate gel from bottom lip.
 * 8) Flip over and transfer gel to staining tray that has been prefilled with 100mL deionized water (see below).
 * 9) *Use lid of a 1000&mu;L pipette tip box.

You have three options for staining at this point.


 * 1) Knight:NuPAGE electrophoresis/Fast staining (fast but less sensitive protocol)
 * 2) Knight:NuPAGE electrophoresis/Slow staining (slow but more sensitive protocol)
 * 3) Knight:NuPAGE electrophoresis/Hybrid staining (allows you to see results immediately but take gel picture when the gel is stained more thoroughly)

See Knight:NuPAGE electrophoresis/Gel drying for instructions on how to dry and preserve the gel.

Marker Sizes

 * The SeeBlue Plus 2 marker (blue unless another color is indicated); 10 μL samples have around 1-2 μg of protein per band
 * Myosin 188 KD (MES buffer) 191 KD (MOPS buffer)
 * Phosphorylase B 98 KD (MES buffer) 97 KD (MOPS buffer) (orange)
 * BSA 62 KD (MES buffer) 64 KD (MOPS buffer)
 * Glutamic dehydrogenase 49 KD (MES buffer) 51 KD (MOPS buffer)
 * Alcohol dehydrogenase 38 KD (MES buffer) 39 KD (MOPS buffer)
 * Carbonic anhydrase 28 KD
 * Myoglobin 17 KD (MES buffer) 19 KD (MOPS buffer) (purple)
 * Lysozyme 14 KD
 * Aprotinin 9 KD
 * Insulin B chain 3 KD


 * Marker chart for Invitrogen Bis-Tris and Tris-Acetate Nupage gels

Safety

 * Use nitrile gloves when handling acrylamide.
 * Dispose of acrylamide gels and trays as hazardous.

See here for detailed safety information.