2.2.2 Gel Preparation

The Gel Consists of Separating Gel and Stacking Gel
The upper layer is the stacking gel, which has a relatively large pore size and no molecular sieve effect. Its pH is 6.8. Due to the high voltage gradient formed by fast and slow ions, denatured protein molecules are compressed into a very thin layer during electrophoresis, significantly improving resolution.
The lower layer is the separating gel, which has smaller pore size and exhibits a molecular sieve effect. Its pH is 8.8. Denatured protein molecules carry essentially the same negative charge, so their migration rate is mainly determined by their molecular weight.
The preparation sequence requires pouring the separating gel first, followed by pouring the stacking gel on top of the separating gel.
Formulations of Gels with Different Concentrations
1. 6% Gel
Solution Component Volume Required for Gels of Different Volumes (ml)    
6% Gel 5ml 10ml 15ml 20ml
H₂O  2.6 5.3 7.9 10.6
30% Acr (Acrylamide Solution)  1 2 3 4
1.5M Tris-HCl (pH 8.8)  1.3 2.5 3.8 5
10% SDS (Sodium Dodecyl Sulfate)  0.05 0.1 0.15 0.2
10% AP (Ammonium Persulfate)  0.05 0.1 0.15 0.2
TEMED (Tetramethylethylenediamine)  0.004 0.008 0.012 0.016
2. 8% Gel
Solution Component Volume Required for Gels of Different Volumes (ml)    
8% Gel 5ml 10ml 15ml 20ml
H₂O  2.3 4.6 6.9 9.3
30% Acr  1.3 2.7 4 5.3
1.5M Tris-HCl (pH 8.8)  1.3 2.5 3.8 5
10% SDS  0.05 0.1 0.15 0.2
10% AP  0.05 0.1 0.15 0.2
TEMED  0.003 0.006 0.009 0.012
3. 10% Gel
Solution Component Volume Required for Gels of Different Volumes (ml)    
10% Gel 5ml 10ml 15ml 20ml
H₂O  1.9 4 5.9 7.9
30% Acr  1.7 3.3 5 6.7
1.5M Tris-HCl (pH 8.8)  1.3 2.5 3.8 5
10% SDS  0.05 0.1 0.15 0.2
10% AP  0.05 0.1 0.15 0.2
TEMED  0.002 0.004 0.006 0.008
4. 12% Gel
Solution Component Volume Required for Gels of Different Volumes (ml)    
12% Gel 5ml 10ml 15ml 20ml
H₂O  1.6 3.3 4.9 6.6
30% Acr  2 4 6 8
1.5M Tris-HCl (pH 8.8)  1.3 2.5 3.8 5
10% SDS  0.05 0.1 0.15 0.2
10% AP  0.05 0.1 0.15 0.2
TEMED  0.002 0.004 0.006 0.008
5. 15% Gel
Solution Component Volume Required for Gels of Different Volumes (ml)    
15% Gel 5ml 10ml 15ml 20ml
H₂O  1.1 2.3 3.4 4.6
30% Acr  2.5 5 7.5 10
1.5M Tris-HCl (pH 8.8)  1.3 2.5 3.8 5
10% SDS  0.05 0.1 0.15 0.2
10% AP  0.05 0.1 0.15 0.2
TEMED  0.002 0.004 0.006 0.008
6. Formulation of Stacking Gel
Solution Component Volume Required for Gels of Different Volumes (ml)    
Stacking Gel 2ml 5ml 8ml 10ml
H₂O  1.4 3.4 5.5 6.8
30% Acr  0.33 0.83 1.3 1.7
1.0M Tris-HCl (pH 6.8)  0.25 0.63 1 1.25
10% SDS  0.02 0.04 0.08 0.1
10% AP  0.02 0.04 0.08 0.1
TEMED  0.002 0.005 0.008 0.01