Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Summary

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.

Abstract

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.

Introduction

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 labeling^1,2,3,4. 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 stain^5,6. In the alkaline ammoniacal s....

Protocol

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.

1. Perform SDS-PAGE with 4% - 12% Bis-Tris protein gels (1 mm, 15-well) using a mini gel tank filled with 2-(N-morpholino)ethanesulfonic acid (MES) buffer.
2. Dilute the samples with a mixture of distilled water, lithium dodecyl sulfate (LDS) buffer, and a sample-reducing agent.
3. Load the first lane with double the amount of stock (10 µL), followed by ....

Results

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.......

Discussion

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

Materials

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