Protocol
Part 1: Preparation from the CBB staining solution
60-80 mg of CBB G-250 are dissolved in 1 liter of bidistilled water by stirring for just two-4 hrs. Finally, 3 ml of concentrated HCl is put into the fast solution with stirring for an additional minute and stored at nighttime for later. The answer could be stored for days as much as several several weeks without losing its staining efficiency.
Concentrated HCl ought to be handled using the usual care und used within fume hood. The ultimate solution is going to be at approximately pH 2, so mitts ought to be used and then any connection with your skin ought to be prevented.
Part 2: SDS-PAGE
Appropriate aliquots of protein samples are combined with loading buffer to some final power of 1x loading buffer. We use 2x loading buffer with 125mM Tris/H3PO4 (pH 7.5 at 25ଌ), 2mM EDTA, 4% SDS, 200mM DTT, .02% bromophenol blue and 50% glycerol. Other loading buffers for SDS-PAGE can be used well.
Protein samples are heated for around 5 min before loading. Meanwhile, the gel electrophoresis chamber is ready for that run. We use precast 4-12% NuPAGE© Bis-Tris gels (Invitrogen) inside a XCell SureLock® Small-Cell (Invitrogen) with MES-buffer because the running buffer but every other gel and electrophoresis system can be used well.
Protein samples are loaded around the gel and electrophoresis run for 50 min at 220V.
Part 3: Staining from the gel
The gel cassette is disassembled and also the gel put into a box for that subsequent washing steps.
About 100 ml of bidistilled water is put into the gel and heated within the micro wave for thirty seconds. Heating ought to be stopped before boiling occurs. This area using the gel will be put on a shaker for several-5 min. which washing step is repeated two times with freshwater.
Enough CBB staining option would be put into cover the gel within the box and also the box heated within the microwave for 10 sec. without boiling. This area using the gel will be put on a shaker for finishing the staining. Already after one minute, protein bands could be observed, after 15-30 min. the staining is powerful enough generally.
The staining option would be put off and 50-100 ml bidistilled water is added to be able to further destain the sunshine blue reputation of the gel on the shaker. Water could be substituted with freshwater for more destaining as needed.
The gel could be scanned, photographed or dried for lengthy-term storage
Part 4: Representative Results:
See Fig. 1 for any correctly stained gel following a described procedure.
See Fig. 2 for any gel that is not washed lengthy enough before staining and residual SDS inhibits efficient staining. Observe that the marker lanes (*) retain the equivalent marker proteins.
Fig. 1: Representative gel stained after loading examples of a protein purification (*: molecular weight marker).
Fig. 2: CBB stained gel that is not washed lengthy enough before CBB staining. The protein bands appear less strong (observe that the marker lane * offers the equivalent protein as with Fig. 1).
Discussion
The washing steps are crucial for the efficient staining from the proteins. A lower washing time below 2 min or reduced amount of water (㱐ml) can lead to pale blue protein bands, probably because of greater levels of residual SDS within the gel.
When the proteins will be analysed by mass spectrometry, the heating stages in the micro wave ought to be skipped, time for laundry from the gel extended to around 10 min in every step and also the staining time extended before the band intensity is powerful enough. Heating the gel leads to crosslinking of proteins towards the gel matrix and therefore recognition from the proteins by mass spectrometry might be hampered.
Rather of CBB G-250, it's possible to use CBB R-250 based on the original protocol by Wondrak. We've not compared both dyes alongside once we were built with a stock of CBB G-250 within our lab coupled with great results with this particular dye.
The rate from the procedure and also the omission of toxic or hazardous solvents within the washing and staining steps are the most crucial and convincing factors for implementing this protocol. The sensitivity is incorporated in the same selection of classical CBB staining protocols and commercial CBB staining solutions and is not a restricting factor for the SDS-PAGE analysis in the area of expression and purification of recombinant proteins.
Disclosures
No conflicting interests. The process described above was initially printed inside a patent application by E.M. Wondrak (see Ref.).
Acknowledgments
We wish to acknowledge the technical aid of Ines Racké.
References
- Wondrak EM, inventor. Process for fast visualization of protein. 6319720 (B1) US patent. 2001
- Wondrak EM, inventor. Solution for fast visualization of protein. 2001046709 (A1) US patent. 2001
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