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Method Article
This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.
Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.
Part 1: Two-dimensional (2-D) gel electrophoresis using cup-loading
Part 2: Colloidal Coomassie staining with CBB G-250
1. Staining solutions
Note: for the preparation of the staining solutions, use chemicals with high quality such as analytical grade (p.a.) and water of high purity as you get from Millipore systems (Milli-Q water).
Coomassie solution: | ad 2000 ml H2O | |
0.02 % (w/v) | CBB G-250 | 0,4 g |
5 % (w/v) | aluminum sulfate-(14-18)-hydrate | 100 g |
10 % (v/v) | ethanol (96%) | 200 ml |
2 % (v/v) | orthophosphoric acid (85 %) | 47 ml |
Note: for the preparation of the staining solution, the sequential addition of the components in the following order has to be maintained:
Note: the final staining solution has a dark green-bluish appearance and is full of particles swimming around:
Destaining solution: | ad 2000 ml H2O | |
10 % (v/v) | ethanol (96 %) | 200 ml |
2 % (v/v) | orthophosphoric acid (85 %) | 47 ml |
2. Staining procedure
Part 3: Representative results
If you follow the protocol described above you will get on your 2-D gel distinctive resolved and well stained dark blue protein spots. Even compared to a 2-D gel stained with silver according to Shevchenko et al.5 ,a protocol claiming good sensitivity and compatibility with mass spectrometry, we achieve equal staining results.
Figure 1: Final outcome of the experimentation described above. Kang's Coomassie protocol (A) performs strongly like the silver staining according to Shevchenko et al. (B) in detecting proteins after 2-D gel electrophoresis.
Part 4: Tips and tricks
Innovative or just another Coomassie protocol?
At the moment there exist multiple protocols for staining procedures with Coomassie Brilliant Blue. Most of them result from minor or major modifications of one of the most commonly used protocols by Neuhoff and colleagues2. Also Kang's protocol based on Neuhoff's formula. But is it really an alternative Coomassie staining method for proteomic research? We will picture two main issues, detection limit and usability, to evidence that it...
I have nothing to disclose.
We thank Dr. Nicola Wiethölter for preparing and staining of 1-D gels.
This work was funded by a grant from the Deutsche Forschungsgemeinschaft (GRK 1089/project 5 to ND and SM) and supported by a research fellowship from the Jürgen Manchot Stiftung to ND.
Name of product | Company | Catalogue No. | Comments |
For IEF and SDS-PAGE: | |||
Immobiline DryStrip gels | GE Healthcare | 17-6001-94 | Can even be used 2 years after expire date. |
Immobiline DryStrip Reswelling Tray | GE Healthcare | 80-6371-84 | Do not clean with organic solvents. Designed for 7-18 cm IPG-strips. |
Immobiline DryStrip Kit | GE Healthcare | 18-1004-30 | Includes a tray, electrode holder, anode and cathode electrodes, aligner and sample cup bar and sample cups. |
EPS 3501 XL Power Supply | GE Healthcare | 18-1130-05 | Supplies voltage up to 3500 V. |
Multiphor II Electrophoresis Unit | GE Healthcare | 18-1018-06 | Movable electrodes enable IEF in IPG strips of all length (7-24 cm IPG strips) |
PerfectBlue gel system Twin S | Peqlab | 45-1010-C | SDS-PAGE in 10x10 cm mini-gel format. Gel chamber includes a cooling system. |
For colloidal Coomassie G-250 staining: | |||
staining dishes with lids | VWR | 216-3412 | Fits for mini-gels. Stackable on shaker. |
aluminium sulfate-18-hydrate | Merck | 1.01102.5000 | We made best experience with Merck. Just available in 5 kg package. |
orthophosphoric acid | Prolabo | 20 624.295 | Sold in glass bottles. |
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