Staining PVDF membranes with CBB R-250 for protein sequencing
Laboratory of Biomolecular Analysis, Institute for Protein Research, Osaka University
2021.10.14 updated
Staining solution
0.1 mg/ml CBB R-250/10 % Methanol/7% Acetic acid
---To make 10 ml staining solution, mix the followings.
0.1 ml
10㎎/ml CBB R-250 in Methanol
1 ml
Methanol
0.7 ml
Acetic acid
8.2 ml
Water
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.
3. Shake in destaining solution for 3~30 min with several solution changes.
4. Rinse in water.
5. Dry in the air.
Note
1) Background staining of PVDF membrane depends on the product type of membrane, CBB concentration in staining solution and methanol concentration in destaining solution. If the background staining is too high, increase the methanol conc. in the destaining solution.
2) Both Immobilon P and PSQ work equally for most proteins. However, Immobilon PSQ has higher binding activity for low molecular weight proteins (<20k) than Immobilon P, while it shows higher background staining. See Example.
Example
PVDF membrane:
Immobilon P (4 x 9 cm, left)
Immobilon PSQ (4 x 9 cm, right)
Transfer buffer:
10 mM CAPS/10% Methanol (pH is to be adjusted at around 11 with NaOH)
