Staining PVDF membranes with CBB R-250 for protein sequencing

Laboratory of Biomolecular Analysis, Institute for Protein Research, Osaka University

2021.10.14 updated

PVDF membrane:

Immobilon P (4 x 9 cm, left)

Immobilon PSQ (4 x 9 cm, right)

Transfer buffer:

10 mM CAPS-NaOH (pH11)/10% Methanol

Transfer conditions:

Semi-dry electrotransfer (150 mA, 60 min)

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.

Staining solution: 0.1 mg/ml CBB R-250/10 % Methanol/7% Acetic acid

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

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Total

10 ml

3. Shake in destaining solution for 3~30 min with several solution changes.

Destaining solution: 50% Methanol/7% Acetic acid

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.