Native PAGE Gel Casting and Electrophoresis

Stock Solutions

1. Acrylamide/Bis-acrylamide Solution (stored at 7-10°C, away from light)
   1. Bring the following to 100 mL with mQ dH₂O
      1. 45 g acrylamide
      2. 3 g bis-acrylamide
2. 4x Gel Buffer
   1. Bring the following to 100 mL with mQ dH₂O
      1. 23.56 g Tris-HCl
   2. Adjust pH to 8.8
3. 1x Electrophoresis Buffer
   1. Bring the following to 1000 mL with mQ dH₂O
      1. 14.42 g glycine
      2. 3.04 g Tris Base
   2. Check pH, do not adjust (should be 8.3)
4. 4x Sample Loading Buffer
   1. Bring the following to 15 mL with mQ dH₂O
      1. 3.72 mL 1 M Tris-HCl, pH 6.8
      2. 0.60 mL 1% bromophenol blue
      3. 7.50 mL glycerol

Gel Casting

1. Rinse, dry, and assemble the gel casting equipment

2. Prepare 10% ammonium persulfate (APS) solution (must be made fresh, right before gel casting)
   1. Weigh 100 mg ammonium persulfate
   2. Dissolve in 1.00 mL mQ dH₂O
3. Prepare resolving gel solution by mixing the following in a 50 mL tube
   1. 4.5 mL acrylamide solution
   2. 5.0 mL 4x gel buffer
   3. Bring to 20 mL with mQ dH₂O
   4. 100 μL 10% APS solution
   5. 15 μL TEMED
   6. Mix solution by inversion
      1. Note - polymerization reaction begins immediately after adding the APS and TEMED
      2. Note - this example is for a 10% resolving gel; adjust acrylamide and mQ dH2O quantities for desired resolving gel strength.

4. Quickly fill ~70% of the gel cassettes with the resolving gel solution, roughly 4 mL per cassette.

5. Load a thin layer of isopropanol on top of the resolving gel solution, and allow ~30 minutes for the gel to form.

6. Remove the isopropanol and rinse with mQ dH₂O.

7. Prepare 4% stacking gel solution by mixing the following in a 15 mL tube
   1. 1.3 mL acrylamide solution
   2. 3.75 mL 4x gel buffer
   3. Bring to 15 mL mQ dH₂O
   4. 50 μL 10% APS solution
   5. 5 μL TEMED
   6. Mix solution by inversion

8. Quickly fill the remaining space in the gel cassettes with the stacking gel solution

9. Carefully insert gel well combs to avoid air bubbles, and allow ~30 minutes for the gel to form

Gel Storage

1. Mix the following in a resealable zipper storage bag
   1. 0.5 mL 4x gel buffer
   2. 1.5 mL mQ dH₂O

2. Place one gel plate per bag with storage buffer

3. Place bags in an air tight storage container

4. Store the container at 4°C

5. Gels should remain viable for about 4 weeks

Sample Preparation

1. Mix protein solution with 4x sample loading buffer in a 3:1 ratio

2. Centrifuge at 17,000 xg for 5 min to remove any solid debris

3. Do not heat the samples like when running denatured PAGE

Electrophoresis

1. Tightly lock the gel into the electrophoresis gasket

2. Load both the inner well and the outer chamber with 1x electrophoresis buffer, and check for gasket leaks

3. Load desired sample volumes (1-5 ug protein will stain adequately) into each well

4. Begin electrophoresis with a constant 50 V until the samples enter the stacking gel

5. Increase to 120 V and allow the gel to run until the loading dye reaches the bottom of the gel

6. Remove gel from gasket, rinse with water, and carefully separate gel plates

7. Return electrophoresis buffer to stock bottle - up to five successive uses before a fresh buffer should be prepared

Important Notes

1. The gel composition in this procedure is only suitable for proteins and peptides with isoelectric points below 8.6

2. For proteins with pI values above 8.6
   1. Buffered gels must be cast with a pH below the sample pI
      1. e.g. 30 mM Histidine buffered gels at pH 6.1 for a basic protein with a pI of 10.4
   2. The direction of electrical current must be flipped, so that positively charged species move towards the bottom of the gel
3. Both gel buffer pH and acrylamide gel density can be varied to improve resolution for each specific protein of interest.