Protein Extraction Buffer (Bakhsh et al.

)
Component

80ml

40ml

5ml

8.4ml

4.2ml

0.525ml

8ml

4ml

0.5ml

5 M NaCl

4.8ml

2.4ml

0.3ml

2 M Tris-Cl
0.7%
Phenylmethylsulphonylfluoride
(PMSF) dissolved in
isopropanol
NH4Cl

0.4ml

0.2ml

0.025ml

8ml

4ml

0.5ml

428mg 214mg 26.75mg

Dithiothreitol (DTT)

140mg

0.5 M EDTA (pH 8.0)

Glycerol

70mg

8.75mg

Protein Extraction Buffer (Rodrigues et al.)

1. Tris-buffered Phenol
2. SDS Buffer
Components with Working
Concentration
0.1 M Tris (pH 8.0)
2% SDS

Stock
Concentrations

0.8ml

8ml

50ml

1.5 M

0.0533

0.533

3.33125

10%

0.16

1.6

10

0.04

0.4

2.5

40 mM

0.0199

0.199

1.24375

48%

0.5

31.25

5% Mercaptoethanol
1 mM PMSF (in isopropanol)
30% Sucrose

3. Ammonium Acetate
0.7708g in absolute methanol (100ml)
4. Acetone (80%)
1. Pulverized samples (250 mg) were resuspended in 0.8 mL of Tris-buffered phenol, pH 8.0,
and 0.8 mL of SDS buffer.
2. The samples were vigorously vortexed for 10 min and centrifuged at 16,000 g for 5 min at 4
C
3. The top phenol layer (0.5 mL) was transferred to a new tube.
4. Proteins were precipitated for 2 h at 20 C with three volumes of pre-cooled 0.1 M
ammonium acetate in absolute methanol.
5. Pelleted by centrifugation (16,000 g for 5 min at 4 C)
6. The pellet was washed once with pre-cooled 0.1 M ammonium acetate in methanol and
once with pre-cooled 80% v/v acetone, followed by vacuum drying.