Western Blotting of RuBPS stained proteins
A. RuBPS staining protocol I (quality)
1. Fix the gel in 30% EtOH, 10% acetic acid overnight
2. Rinse the gel in 20% EtOH for 30 min and repeat 3 times
3. Incubate the gel in 1 mM RuBPS solution for 6 h
4. Equilibrate the gel in water for 10 min and repeat once
5. Destain the gel with 40% EtOH/10% acetic acid for 15 h
6. Equilibrate the gel in water for 10 min repeat once and scan
all% are in V/V
Procedure as published in Proteomics 2004, 4, 599–608.
B. Western Blotting
1. Equilibrate RuBPS stained 2-D gels, SDS gels, PVDF and
nitrocellulose membranes were equilibrated in a blotting buffer (50 mM
boric acid, pH 9.0) for 30 min.
2. Transfer the stained proteins to a nitrocellulose membrane (or a stack
of up to 8 membranes) for 45 min. at 200 mA and 500 V or to a PVDF
membrane for 90 min. at 200 mA and 500 V under cooling. Scan the
3. Wash the membrane(s) twice with TBS (10 mM Tris/HCl pH 8.0, 150
mM NaCl) and saturated with TBS containing 1% w/v milk powder for
4. Incubate the membrane(s) overnight with a the antibody I (specific
antibody against the protein you want to detect) (1:5000) in TBS
containing 0.5% w/v BSA.
5. Rinse short with TBS
6. Incubate the membrane(s) with antibody II (antibody against antibody
I) (1:5000) in TBS, 0.5% w/v BSA for 2 h.
7. After the reaction stopped, wash the membrane twice in 0.5% TBS for
8. Treat the membrane(s) with peroxidase staining solution (TBS, 6% v/v
chloro-1-naphthol 0.3% w/v in MeOH, 0.002% v/v H2O2 30%) for 2 to
9. Store the membrane(s) in 0.5% TBS and scan again
Procedure as described http://www.ruthenium.ag.vu/western_blotting_.html
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