Bidirectional gel fluorescence staining and imaging experiments
Staining of biphasic gel electrophoresis is a crucial step in comparative proteomics studies regarding the number of proteins to be measured, the accurate quantification of protein spots, and their compatibility with mass spectrometry techniques. This chapter describes a variety of dyes for gel staining that are compatible with mass spectrometry techniques: Kaumas Brilliant Blue, Silver Nitrate, Sypro Ruby, Deep Purple.This experiment was derived from the "Guide to Plant Proteomics Experiments" [French] H. Tillament, M. Zivi, C. Damerweil, and V. Mitschine, eds.
Operation method
Bidirectional gel fluorescence staining and imaging
Materials and Instruments
Extraction solution Wash solution Dissolving solution First direction electrophoresis buffer Reduction solution Alkylation solution Agarose solution Acrylamide gel solution Move 3.1 Extraction method for total soluble protein (TCA/acetone method) For more product details, please visit Aladdin Scientific website.
Bidirectional gel electrophoresis Optical scanner or densitometer
( 1 ) Cells were collected from Arabidopsis cell suspension culture.
( 2 ) Grind the cells in liquid nitrogen.
( 3 ) Add the extract to the ground cell powder and place at -20 °C for at least 30 min (see Note 7).
( 4 ) Centrifuge at 42000 g to remove insoluble material and wash three times with Wash Solution (see Note 8) (see Note 8).
( 5 ) Dry the tubes in air.
( 6 ) Dissolve the proteins by shaking and settling in the dissolution solution for 2 h (see Note 9).
( 7 ) Determine the protein concentration according to the Bradford method [ 3, 4], using an aliquot of the sample and serum albumin as a standard curve (aliquots are solutions of the same proportion).
3.2 Bidirectional gel electrophoresis
( 1 ) Directly hydrate the IPG gel (18 cm, pH 4.0~7.0) with 100 μg of protein solution matched with the first direction electrophoresis solution.
( 2 ) Perform isoelectric focusing up to 100 kV/h.
( 3 ) Prior to the second direction electrophoresis, the proteins were reduced in the reduction solution and then alkylated in the alkylation solution for 15 min each.
( 4 ) The strips were embedded in agarose solution on top of 11% acrylamide gel.
( 5 ) Perform SDS-PAGE in electrophoresis buffer at 15 mA per gel, overnight at 10°C (see Note 10).
3.3 CCB Staining and Imaging (see Note 11, Note 12)
Gels are stained according to the method of Neuhoff et al. For each step, use 300 ml of solution per gel. All operations require shaking.
( 1 ) Submerge the gel in fixative for at least 2 h or overnight to fix the protein.
( 2 ) Wash the gel with water three times for a total of 30 min.
( 3 ) Transfer the gel to incubation solution for 1 h (see Note 13) and then to staining solution (see Note 13, Note 14).
( 4 ) Develop the gel in the staining solution for 5 days with gentle shaking.
( 5 ) Wash the gel with water and acquire an image with a scanner at 300 dpi resolution (Fig. 14-1). 
3.4 SN Staining and Imaging (see Note 11, Note 12)
Gels are stained according to the method of Mortz et al [15]. For each step, 300 ml of solution per gel is used. All operations require shaking.
( 1 ) Submerge the gel in fixative for at least 2 min or overnight to fix proteins.
( 2 ) Wash the gel 3 times for a total of 20 minutes in the Wash Solution.
( 3 ) Soak the gel for a short time (2 min) in sensitizing solution (see Note 15).
( 4 ) Wash the gel with water 3 times for 5 min.
( 5 ) Soak the gel in staining solution for 20 min.
( 6 ) Rapidly wash the gel with water twice for 1 min.
( 7 ) Place the gel in the color development solution for 5~10 min (see Note 16).
( 8 ) Terminate the color development in the termination solution for 5 min and transfer the gel to the storage solution.
( 9 ) Use a scanner to obtain an image at 300 dpi resolution (Figure 14-1).
3.5 SR Staining and Imaging (see Note 11, Note 12, and Note 17)
For each step, use 300 ml of solution per gel. All operations require shaking.
( 1 ) Submerge the gel in fixative for 30 min to fix the protein.
( 2 ) Stain the gel in the staining solution for at least 30 min (maximum 1 h).
( 3 ) Wash the gel with washing solution for 30 min.
( 4 ) Images were acquired using a FLA-5000 analyzer with 473 nm laser excitation and a long channel filter Y510. The selection conditions were 100 μm resolution, 16-bit grayscale, and photocell voltage of 700 V (Figure 14-2). 
3.6 DP Staining and Imaging (see Notes 11, 12, and 17)
For each step, use 300 ml of solution per gel. All operations require shaking.
( 1 ) Submerge the gel in fixative for 1 h to fix the protein.
( 2 ) Wash the gel with water 4 times for 10 min each time.
( 3 ) Submerge the gel in staining solution for 1 hour.
( 4 ) Wash the gel with washing solution 1 twice for 10 min, and then wash it with washing solution 2 times for 10 min.
( 5 ) Images were acquired using a FLA-5000 analyzer with 532 nm laser excitation and long channel filter 0575. The selection conditions were 100 μm resolution, 16-bit grayscale, and photocell voltage of 700 V (Fig. 14-2).
3.7 C16-F Staining and Imaging (see Notes 10, 11, 17, and 18)
Gels are stained according to the method of Kang et al [27]. For each step, use 300 ml of solution per gel. All operations require shaking.
( 1 ) Proteins are fixed and stained simultaneously by submerging the gel in fixative and staining solution.
( 2 ) Wash the gel at least twice for 5 min each with Wash Solution.
( 3 ) Acquire images using a FLA-5000 analyzer with 473 nm laser excitation and a long channel filter Y510, with a 100 μm resolution, 16-bit grayscale, and a photocell voltage of 700 V (Figure 14-2).
3.8 Comparison of Dyes
In the above staining methods, when the sample was 100 μg of total soluble proteins extracted from Arabidopsis thaliana cultured cells, 250 protein spots were usually detected by CCB, 450 protein spots by C16-F, 550 protein spots by DP, 600 protein spots by SN, and 800 protein spots by SR (Fig. 14-1 and Fig. 14-2). The main characteristics of these methods are summarized in Table 14-1. Staining methods were compared according to dye type, staining step, and operation time. Image quality was evaluated by background effect, spot saturation, and dye sensitivity. 