A subscription to JoVE is required to view this content. Sign in or start your free trial.
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 s....
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.
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
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 |
This article has been published
Video Coming Soon
ABOUT JoVE
Copyright © 2024 MyJoVE Corporation. All rights reserved