Tris/Tricine/SDS/Urea Page Gel Casting and Electrophoresis

Stock Solutions

  1. Acrylamide/Bisacrylamide Solution (Store at 7-10°C)
    1. Bring the following to 100 ml with mQ dH2O
      1. 48 g acrylamide
      2. 3 g bis-acrylamide
  2. 3x Gel Casting Buffer
    1. Bring the following to 1000 ml with mQ dH2O
      1. 158.4 g Tris-HCL
      2. 241.8 g Tris Base
      3. 3.0 g SDS
    2. Adjust pH → 8.45
  3. 10x Cathode Buffer
    1. Bring the following to 1000 ml with mQ dH2O
      1. 121.1 g Tris Base
      2. 179.2 g Tricine
      3. 1.0 g SDS
    2. Check pH - Do Not Adjust - Should be ~8.25
  4. 10x Anode Buffer
    1. Bring the following to 1000 ml with mQ dH2O
      1. 19.2 g Tris-HCl
      2. 106.4 g Tris Base
    2. Adjust pH → 8.9
  5. 5x Sample Loading Buffer (w/o reductant)
    1. Bring the following to 5 ml with mQ dH2O
      1. 2 ml 0.5 M Tris-HCl pH 6.8
      2. 250 μl 1% Bromophenol Blue
      3. 1 ml glycerol (ρ = 1.25 g/ml)
      4. 500 mg SDS
  6. 2x Sample Loading Buffer (with reductant and denaturant)
    1. Bring the following to 10 ml with mQ dH2O
      1. 4.8 g urea
      2. 4 ml 5x sample loading buffer
      3. 100 μl β-mercaptoethanol

Gel Casting Procedure

  1. Construct gel casting apparatus

  2. Prepare 10% APS - Combine the following
    1. 100 mg APS
    2. 1.0 ml mQ dH2O
  3. Prepare 16% gel solution
    1. Combine the following in a 15 ml disposable tube
      1. 5.4 g urea
      2. 5 ml gel casting buffer
      3. 5 ml acrylamide/bis-acrylamide solution
    2. Lightly agitate to fully dissolve urea (tilt table of shaker)
    3. Bring to 15 ml with mQ dH2O
    4. Add the following to initiate solidification
      1. 60 μl 10% APS
      2. 6 μl TEMED
  4. Immediately Dispense 3.5 ml 16% resolving solution (3) into gel casting apparatus

  5. Dispense a layer of isopropanol on the top of gel solution and allow ~30 min for solidification

  6. Pour off isopropanol and rinse the well out with mQ dH2O

  7. Prepare 10% gel solution
    1. Combine the following in a 15 ml disposable tube
      1. 5.4 g urea
      2. 5 ml gel casting buffer
      3. 3 ml acrylamide/bis-acrylamide solution
    2. Lightly agitate to fully dissolve urea (tilt table of shaker)
    3. Bring to 15 ml with mQ dH2O
    4. Add the following to initiate solidification
      1. 75 μl 10% APS
      2. 7.5 μl TEMED
  8. Immediately dispense 2 ml 10% gel solution (7) into gel casting apparatus

  9. Dispense a layer of isopropanol on the top of gel solution and allow ~30 min for solidification

  10. Pour off isopropanol and rinse the well out with mQ dH2O

  11. Prepare 4% stacking solution 1. Combine the following in a 15 ml disposable tube
    1. 8 ml mQ dH2O
    2. 3 ml gel casting buffer
    3. 1 ml acrylamide/bis-acrylamide solution 2. Add the following to initiate solidification
    4. 100 μl 10% APS
    5. 10 μl TEMED
  12. Immediately dispense 1.5 ml 4% stacking solution into gel casting apparatus and immediatly insert well comb

  13. Allow ~30 min for solidification

  14. Store gels in resealable zipper bags containing 0.5 mL 3x gel casting buffer and 1.0 mL mQ dH2O at 4 °C

Sample Preparation

  1. Protein samples
    1. Combine the following in a 1:1 ratio to desired volume (Final protein mass to should be at 1-4 μg/band)
      1. Protein sample (Excessive salt and lipids can cause extreme distortions)
      2. 2x Sample loading buffer
    2. For most proteins, place samples in 95 C bath for 5 min. (Incubate in 40 C for up to an hour if to avoid irreversible protein aggregation of membrane proteins if necessary)
    3. Centrifuge at 17,000 xg for 5 minutes

Electrophoresis Procedure

  1. Position gel in eletrophoresis apparatus. (Ensure proper alignment of rubber seal overhang to the lip of the inner glass plate to avoid leakage)

  2. Fill inner well with 1x anode buffer

  3. Inject samples into respective wells (~10ul max in 15-well gel and 20ul max in 10-well gels) with a final mass of 1-4 ug per expected band

  4. Fill outer well with 1x cathode buffer

  5. Apply 30 V until sample has completely entered the stacking gel

  6. Increase to 100 V until the sample buffer dye front has reached the bottom of the gel

  7. Carefully remove gel cassette, separate plates under flowing water and carefully extract gel

  8. Submerge gel in fixing solution

  9. Discard cathode buffer and return anode buffer to stock bottle.


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