Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Summary

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.

Abstract

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.

Protocol

Part 1: Two-dimensional (2-D) gel electrophoresis using cup-loading

1. IPG-strip rehydration and isoelectric focusing (IEF)
   • Rehydrate Immobiline DryStrip gels, pH 6-11 (7 cm) in 125 μl rehydration solution [7 M urea, 2 M thiourea, 4% CHAPS, 50 mM hydroxyethyldisulfide and 2% IPG Buffer pH 6-11] using Immobiline DryStrip Reswelling Tray for at least 10 hours.
   • Dissolve precipitated protein sample in IEF sample buffer [7 M urea, 2 M thiourea, 2% CHAPS, 2% ASB-14, 50 mM hydroxyethyldisulfide and 2% IPG Buffer pH 6-11] corresponding to 60-100 μg protein per 100 μl.
   • After solubilization (at least 30 minutes at room temperature), apply....

Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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.
staining dishes with lids	VWR international	216-3412	Fits for mini-gels. Stackable on shaker.
aluminium sulfate-18-hydrate	Merck & Co., Inc.	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.