Protokolle:

1.RuBPS staining protocol I (quality):

RuBPS staining protocol II (fast):

RuBPS staining protocol III (co-electrophoretical)

RuBPS staining protocol IV (loading buffer

Preparation of RuBPS staining solution

• Preparation of the staining solution: Dissolve 5 mg of RuBPS (one unit) in 3L of deionised water to have a 1 mM RuBPS staining solution.
• Preparation of the staining solution from a stock solution: Dissolve 5 mg of RuBPS (one unit) in 150 ml of deionised water to have a 20 mM stock solution.Dissolve 50 ml of the 20 mM stock solution to have a 1 mM RuBPS staining solution

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 10min
• 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 3min
• 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.

FAQs about RuBPS:

• 1. How many gels can be stained with one unit of RuBPS and what are the costs per gel? From one unit RuBPS is sufficient to prepare 3 litres of staining solution. 3 L of staining solution is enough to stain 15 large (24cm) 2D-gels or 60 small SDS gels.This means that RuBPS is about 20 times cheaper than other comparable dyes. RuBPS has absolutely no competitor concerning the costs.
• 2. Is RuBPS difficult to handle? No it isn´t. You just open the vial, dissolve the RuBPS powder in 3 L of water, that´s it
• 3. What are the shipping costs? We sell the only dye and not water! Shipping milligrams of dye is much cheaper than shipping tons of water as most our competitors do. So it easy to understand that shipping milligrams of dye saves your time and your money! So the shipping costs a as low as (Germany 5EUR)
• 4. What is the stability of RuBPS? RuBPS is sold as solid powder. In this physical form RuBPS is rock stable for years without cooling.As soon as it is dissolved to 20 mM, RuBPS is stable at least for 5 Years is stored in a dark bottle at 4°C (normal fridge).
• 5. How long does it take to stain a gel with RuBPS? The staining of one gel with RuBPS will take about 1 hour. So it is as fast as CBB staining but much more sensitive with a much broader linear dynamic range.
• 6. Is RuBPS compatible with MS? Yes it is. It is equal to CBB but has a 4-times increased sensitivity and a much broader linear dynamic range.
• 7. Is RuBPS compatible with blotting procedures? Yes it is. We sell you a dye for gel- and blott-staining. Just stain your gel with RuBPS, scan it, and then do the transfer to the membrane (nitrocellulose or PVDF). Scan the membrane, and you receive a copy of your gel on the membrane. RuBPS does not interfere with the immunodetection! So you can detect all protein spots in the gel and on the membrane copy and specifically label your target protein at once! No other dye can do this. For the same purpose our competitors will sell you two staining solutions, which mainly contain water, at a 40 times higher price. As solutions are heavy they cause high shipping costs and take you a lot of space for storage. There is no need to buy two dyes to stain membranes and gels, but our competitors simply think:why only sell one, if you could sell two for the double price?.
• 8. Is RuBPS a copy of SYPRO Ruby? The answer is no. There were always rumours that RuBPS is a copy of SYPRO Ruby, but as it is well known, it is not The rumour originally came from the Rabilloud article in 2000 where he started these speculations. This rumour was boosted when RuBPS was called “Fluka-SYPRO” by some speakers at the Proteomic Forum 2003 in Munich, Germany. Frequently RuBPS has been called “Home made SYPRO” or “Home brew Version of SYPRO Ruby” which is simply not true. All these rumours are nothing else than false. RuBPS is not SYPRO and was never intended to be SYPRO or to imitate SYPRO.
• 9. What is the meaning of RuBPS? RuBPS is the short form of Ruthenium (II) tris (bathophenantroline disulfonate) also termed as Ruthenium (II) tris (4,7-diphenyl-1,10-phenantrolin disulfonate).
• 10. What is RuBPS? RuBPS is the transition metal complex Ruthenium (II) tris (4,7-diphenyl-1,10-phenantrolin disulfonate).
• 11. Who invented RuBPS? RuBPS was first synthesized as a precursor molecule for a dye used as a non-radioactive label for oligo nucleotides by Prof. Willi Bannwarth (University of Freiburg i.Br. Germany) while he worked at Hoffmann-La Roche in the 80ies. This was published in: Bannwarth W, Schmidt D, Stallard RL, Hornung C, Knorr R, Müller F:Bathophenantroline-rutheium (II) complexes as nonradioactive labels for dideoxy DNA sequencing which can be measured by time-resolved fluorescence techniques. Helv Chim Acta 1988,71:2085-2099.
• 12. Who developed the staining techniques? Prof. Thierry Rabilloud introduced RuBPS as a fluorescent label for protein detection in polyacrylamide gels. Rabilloud T, Strub JM, Luche S, Girardet JL, van Dorsselaer A, Lunardi J: Ruthenium II tris (bathophenanthroline disulfonate), a powerful fluorescent stain for detection of proteins in gel with minimal interference in subsequent mass spectrometry analysis. Proteome 2000,1:1-13 His initial staining technique was further developed during 6 years in our Lab and published here: Lamanda A, Zahn A, Roder D, Langen H: Improved Ruthenium II tris (bathophenantroline disulfonate) staining and destaining protocol for a better signal-to-background ratio and improved baseline resolution. Proteomics 2004,4:599-608 and here: Lamanda A, Cheaib Z, Turgut MD: Protein buffering in model systems and in whole human saliva. PLoS ONE 2007,Feb 28,2(2):e263. The Methods were first licenced to Sigma-Aldrich and later commercialised.
• 13. How popular is the RuBPS staining method? The RuBPS staining method has been cited in more than 60 scientific articles from plant proteomics to brain research and was taken up as method in 4 Books, was subject to research in 5 PhD thesis and has found its way into wikipedia and arxive. The RuBPS staining method has won the 1st price for R&D in 2002 (DC Bank Avard,Switzerland) and the Wrigley Prophylaxe Avard in Germany for R&D in 2006.