Staining a PVDF membrane with CBB R-250 for protein sequencing
Laboratory of Biomolecular Analysis, Institute for Protein Research, Osaka University
PVDF membrane:
Immobilon P (4 x 9 cm, left)
Immobilon PSQ (4 x 9 cm, right)
Transfer:
Semi-dry electrotransfer (150 mA, 60 min)
with 10 mM CAPS-NaOH (pH11)/10% Methanol
Staining solution:
0.1 mg/ml CBB R-250/10 % Methanol/7% Acetic acid
Destaining solution:
50% Methanol/7% Acetic acid
Protocol
1. After SDS-PAGE and electroblotting, rinse the PVDF membrane with water.
2. Shake the membrane in staining solution for 3 min at room temp.
To make 10 ml of staining solution, mix the following solutions.
| 10 ㎎/ml CBB R-250 in Methanol | 0.1 ml |
| Methanol | 1.0 ml |
| Acetic acid | 0.7 ml |
| Water | 8.2 ml |
3. Shake in destaining solution for 3~30 min with several solution changes.
4. Rinse in water.
5. Dry in the air.
Notes
1) If the background staining is too high, increase the methanol conc. in the destaining solution.
2) Immobilon P and PSQ work equally for most proteins. However, Immobilon PSQ has higher binding activity for low molecular weight proteins (<20k), while it shows higher background staining.
