Tris/Tricine/SDS/Urea Page Gel Casting and Electrophoresis
Stock Solutions
- Acrylamide/Bisacrylamide Solution (Store at 7-10°C)
- Bring the following to 100 ml with mQ dH2O
- 48 g acrylamide
- 3 g bis-acrylamide
- Bring the following to 100 ml with mQ dH2O
- 3x Gel Casting Buffer
- Bring the following to 1000 ml with mQ dH2O
- 158.4 g Tris-HCL
- 241.8 g Tris Base
- 3.0 g SDS
- Adjust pH → 8.45
- Bring the following to 1000 ml with mQ dH2O
- 10x Cathode Buffer
- Bring the following to 1000 ml with mQ dH2O
- 121.1 g Tris Base
- 179.2 g Tricine
- 1.0 g SDS
- Check pH - Do Not Adjust - Should be ~8.25
- Bring the following to 1000 ml with mQ dH2O
- 10x Anode Buffer
- Bring the following to 1000 ml with mQ dH2O
- 19.2 g Tris-HCl
- 106.4 g Tris Base
- Adjust pH → 8.9
- Bring the following to 1000 ml with mQ dH2O
- 5x Sample Loading Buffer (w/o reductant)
- Bring the following to 5 ml with mQ dH2O
- 2 ml 0.5 M Tris-HCl pH 6.8
- 250 μl 1% Bromophenol Blue
- 1 ml glycerol (ρ = 1.25 g/ml)
- 500 mg SDS
- Bring the following to 5 ml with mQ dH2O
- 2x Sample Loading Buffer (with reductant and denaturant)
- Bring the following to 10 ml with mQ dH2O
- 4.8 g urea
- 4 ml 5x sample loading buffer
- 100 μl β-mercaptoethanol
- Bring the following to 10 ml with mQ dH2O
Gel Casting Procedure
-
Construct gel casting apparatus
- Prepare 10% APS - Combine the following
- 100 mg APS
- 1.0 ml mQ dH2O
- Prepare 16% gel solution
- Combine the following in a 15 ml disposable tube
- 5.4 g urea
- 5 ml gel casting buffer
- 5 ml acrylamide/bis-acrylamide solution
- Lightly agitate to fully dissolve urea (tilt table of shaker)
- Bring to 15 ml with mQ dH2O
- Add the following to initiate solidification
- 60 μl 10% APS
- 6 μl TEMED
- Combine the following in a 15 ml disposable tube
-
Immediately Dispense 3.5 ml 16% resolving solution (3) into gel casting apparatus
-
Dispense a layer of isopropanol on the top of gel solution and allow ~30 min for solidification
-
Pour off isopropanol and rinse the well out with mQ dH2O
- Prepare 10% gel solution
- Combine the following in a 15 ml disposable tube
- 5.4 g urea
- 5 ml gel casting buffer
- 3 ml acrylamide/bis-acrylamide solution
- Lightly agitate to fully dissolve urea (tilt table of shaker)
- Bring to 15 ml with mQ dH2O
- Add the following to initiate solidification
- 75 μl 10% APS
- 7.5 μl TEMED
- Combine the following in a 15 ml disposable tube
-
Immediately dispense 2 ml 10% gel solution (7) into gel casting apparatus
-
Dispense a layer of isopropanol on the top of gel solution and allow ~30 min for solidification
-
Pour off isopropanol and rinse the well out with mQ dH2O
- Prepare 4% stacking solution
1. Combine the following in a 15 ml disposable tube
- 8 ml mQ dH2O
- 3 ml gel casting buffer
- 1 ml acrylamide/bis-acrylamide solution 2. Add the following to initiate solidification
- 100 μl 10% APS
- 10 μl TEMED
-
Immediately dispense 1.5 ml 4% stacking solution into gel casting apparatus and immediatly insert well comb
-
Allow ~30 min for solidification
- 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
- Protein samples
- Combine the following in a 1:1 ratio to desired volume (Final protein
mass to should be at 1-4 μg/band)
- Protein sample (Excessive salt and lipids can cause extreme distortions)
- 2x Sample loading buffer
- 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)
- Centrifuge at 17,000 xg for 5 minutes
- Combine the following in a 1:1 ratio to desired volume (Final protein
mass to should be at 1-4 μg/band)
Electrophoresis Procedure
-
Position gel in eletrophoresis apparatus. (Ensure proper alignment of rubber seal overhang to the lip of the inner glass plate to avoid leakage)
-
Fill inner well with 1x anode buffer
-
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
-
Fill outer well with 1x cathode buffer
-
Apply 30 V until sample has completely entered the stacking gel
-
Increase to 100 V until the sample buffer dye front has reached the bottom of the gel
-
Carefully remove gel cassette, separate plates under flowing water and carefully extract gel
-
Submerge gel in fixing solution
-
Discard cathode buffer and return anode buffer to stock bottle.