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Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.
Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.
Many staining methods have been used to visualize proteins following gel electrophoresis, for example using colorimetric dyes such as Coomassie Brilliant Blue, silver stain, fluorescence, or radioactive labeling1,2,3,4. Silver staining is considered to be one of the most sensitive techniques for protein detection requiring simple and cheap reagents. It can be categorized into two major families: the ammoniacal silver stain and the silver nitrate stain5,6. In the alkaline ammoniacal silver method, the silver-diamine complex is produced with ammonia and sodium hydroxide and reduced to metallic silver during development using an acidic formaldehyde solution. The stain accommodates efficiently for basic proteins but shows a compromised performance for acidic and neutral proteins and is, furthermore, limited to classical glycine and taurine electrophoretic systems. In comparison, the silver nitrate stains exploit the high bio-affinity of silver ions to protein, primarily the sulfhydryl and carboxyl groups from the side chains, and tend to stain acidic proteins more efficiently7. After silver ion binding, a developing solution (typically made of a metal carbonate solution containing formaldehyde and sodium thiosulphate) is applied to reduce silver ions to metallic silver grains, which build up a brown-to-dark color to visualize the protein bands.
Although silver staining has been well known for its versatility and high sensitivity since its development in the 1970s8, the method is frequently regarded as tricky. Silver-staining methods have time-restricted steps and show low reproducibility. Since the color of silver stain is usually not uniform and dependent on the reduction step, which is hard to control, the silver stain is not a quantitative method and, thus, not recommended for gel comparison study and protein quantification9. Methods optimized in sensitivity may utilize aldehydes which can also provide a more uniform staining10. However, this is at the expense of further downstream analysis due to the crosslinking of proteins by aldehydes. Fast protocols mostly combine or shorten steps to reduce time, compromising the reproducibility and uniformity of the stain5. As a result, there are numerous silver staining variants within protein gel staining, each optimized to suit certain requirements; for example, simplicity, sensitivity, or peptide recovery rate for downstream analysis. These attributes may also have an impact on each other, and satisfying all requirements in one protocol can be difficult.
In this work, we introduce a new fluorescent silver staining method for protein detection in polyacrylamide gel. In this method, we use a fluorogenic probe for silver ions, TPE-4TA (Figure 1), to visualize the silver-impregnated proteins11. TPE-4TA is designed by the aggregation-induced emission (AIE) principle. It is non-emissive when dissolved in aqueous solution, but is highly emissive in the presence of silver ions. By replacing the chromogenic development in traditional silver stains with a fluorogenic developing step, the fluorescent silver method enables the robust staining of total proteins with a reduced background.
Furthermore, the fluorescent silver stain showed a good dynamic linear range for protein quantification, which is comparable with the widely-used SYPRO Ruby stain and not achievable with traditional silver stains. The gel can be imaged on commonly used gel documentation systems with an ultraviolet lamp (excitation wavelength: 302/365 nm channel; emission: ~490 - 530 nm) at many biological labs.
1. Preparation of the Gel
Note: The demonstration follows a standard protocol to prepare the gel for staining shortly after SDS-PAGE12. In brief, the following steps describe the preparation of the samples and gel electrophoresis.
2. Fixation of the Gel
3. Preparation of the AgNO3 Solution and Silver Impregnation of the Gel
4. Fluorogenic Development of the Gel
5. Destaining and Imaging
The protein bands stained by the fluorescent silver stain exhibit an intense green fluorescence under a 365 nm UV lamp. All 14 protein bands (10 - 200 kDa), from top to bottom, were clearly visible, correlating well with the 14 red-colored ones stained by the SYPRO Ruby dye (Figure 2)10.
Regarding quantitative protein detection, the gels were imaged by a gel imaging system us...
Presented here is a novel fluorescent silver staining method for proteins in polyacrylamide gels. This strategy integrates conventional silver stains and fluorescent stains. The staining exploits the selective binding of silver ion to proteins as in other silver stains but employs a highly sensitive fluorogenic silver probe TPE-4TA to light up the silver bound proteins. Since the fluorogenic probe TPE-4TA can sense silver ions at a fairly low concentration in the nanomolar range
A patent application on this fluorescent silver staining has been filed.
The authors would like to thank Patrick Chan at the Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, for his technical support. S. X. is grateful for the support from the Swedish Research Council (Grant No. 2017-06344).
Name | Company | Catalog Number | Comments |
LDS Sample Buffer (4X) | Thermo Fisher Scientific | NP0007 | Reagent |
4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well | Thermo Fisher Scientific | NP0323BOX | Precast gel |
Sample Reducing Agent (10X) | Thermo Fisher Scientific | NP0004 | Reagent |
MES SDS Running Buffer (20X) | Thermo Fisher Scientific | NP0002 | Reagent |
Mini Gel Tank | Thermo Fisher Scientific | A25977 | Equipment |
300W Power Supply (230 VAC) | Thermo Fisher Scientific | PS0301 | Equipment |
Unstained Protein Ladder | Thermo Fisher Scientific | 26614 | Sample |
Silver nitrate | Sigma-Aldrich | 31630-25G-R | Reagent |
Ethanol | Bragg and co. | 42520J | Reagent |
Acetic acid | J.T. Baker | 103201A | Reagent |
Milli-Q Synthesis A10 | Merk | - | Provides 18.2 MΩ.cm water |
gel documentation system (c600 model) | Azure biosystems | - | Equipment |
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