RuBPS Staining - RuBPS - RuBPS Western Procedure

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

    membrane.

 

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

   10 min.

 

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

    3 min.

 

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

    5 min.

 

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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