BUFFER PREPARATION FOR LAB 1: PROTEIN ISOLATION AND SDS-PAGE

You are required to prepare these solutions and buffers for your experiment. Calculate the
amount needed for each chemical for these buffer solutions.

1M Tris-HCl pH7.5 (used old ones, but need to re-pH)

Final amount: 10mL
MW Final concentration Amount
Tris-Base 121.14 1M

HCl pH7.5

Final amount: 50mL

General Lysis buffer Amount
Tris-HCl pH 7.5 50 mM

NaCl 100 mM

DTT 5 mM

EDTA 1 mM
Monomer stock solution (30% acrylamide, 0.8% N,N’-methylenebisacrylamide)
Final amount: 75mL
Final concentration Amount
Acrylamide 30%

N,N’-methylenebisacrylamide 0.8 %

1M Tris-HCl pH8.8 (used old ones, but need to re-pH)

Final amount: 10mL
MW Final concentration Amount
Tris-Base 121.14 1M

HCl pH8.8

4× resolving buffer solution (1.5M Trisbase pH8.8)

Final amount: 100mL
MW Final concentration Amount
Tris Base 121.14 1.5 M

HCl pH 8.8
1M Tris-HCl pH6.8 (used old ones, but need to re-pH)
Final amount: 10mL
MW Final concentration Amount
Tris-Base 121.14 1M

HCl pH6.8

Stacking Buffer (0.5M Tris Base pH 6.8)

Final amount: 100mL
MW Final concentration Amount
Tris Base 121.14 0.5 M

HCl Adjust to pH 8.8

10% SDS solution

Final amount: 20mL
Final concentration Amount
SDS 10%

10% Ammonium persulfate (APS) buffer

Final amount: 20mL
Final concentration Amount
APS 10%
5× SDS loading buffer (10mM DTT, 0.2M Tris-HCl, 20%Glycerol, 10% SDS, 0.05%
bromophenol blue)
Final amount: 10mL
MW Final concentration Amount
SDS 10%

Glycerol 20%

Tris-HCl (1M) 0.2M

DTT 154.253 10mM

Bromophenol blue 0.05%

10× Laemmli SDS electrophoresis buffer (250mM Trisbase, 1.92M Glycine, 1%SDS)
Total: 200mL
MW Final concentration Amount
Trisbase 121.14 250 mM

Glycine 75.066 1.92 M

 SDS 1%

Fixing Solution
Total: 2L
MW Final concentration Amount
Methanol 50%

Glacial Acetic acid 10%

Staining Solution
Total: 2 L
MW Final concentration Amount
Methanol 45%

Glacial Acetic acid 10%

Coomassie R-250 3g/L

Destain Solution
Total: 2L
MW Final concentration Amount
Methanol 5%

Glacial Acetic acid 7.5%