Electrophoresis image analysis

Figure 3.1. Protein expression mapping using 2-D electrophoresis and mass spectrometry. The purpose is to compare protein expression patterns between cell types or in the same cell type under different growth conditions. Proteins are extracted from the different cell types and separated by 2D gel electrophoresis. Image analysis programs are used to compare the spot intensities between gels and identify proteins that are differentially expressed. The protein of interest is excised from the gel and its identity is determined by mass spectrometry. The power of the method increases greatly if the identity of a large number of proteins on the gel is known and present in a database because information can then be obtained without further mass spectrometry.

Figure 4.1 The comet assay. A single-cell suspension is embedded in agarose on a slide. Cells are then subject to lysis followed by electrophoresis. If present, damaged DNA migrates out of the nucleoid structure during electrophoresis to producing a characteristic comet shape. Double-strand breaks are revealed under neutral conditions, whereas alkali conditions additionally show single-strand breaks and alkali labile sites. Image analysis of stained DNA is used to quantitate the amount of damaged DNA in the comet tail.

In summary, we have the following steps (1) cell suspension, preparation, viability, and density (2) cells in agarose gel on slides (3) lysis (4) DNA unwinding, electrophoresis, neutralization, and staining with a fluorophore (5) image analysis and scoring. 

Horgan G, Glasbey CA. Uses of digital image analysis in electrophoresis. Electrophoresis 1995 16 298-305. 

High resolution two-dimensional electrophoresis with computerized image analysis and data reduction is the highest resolution method currently available for the analysis of complex protein mixture. In this discussion we review the potential of the method for the analysis of agricultural food products including milk, meat products, and wheat, and present one representative study on the analysis of Newton wheat for electrophoretic variants. 

Figure 9. 2D gel image analysis, western immunoblot analysis with anti-CyPA antibody, and immunoelectron microscopic analysis. (A) After 2D gel electrophoresis and protein visualization by silver staining, the 2D gel images of subtiiisin-untreated HIV-1lav-i preparation, subtilisin-treated HIV-1lav-i preparation, and microvesicles were expanded. The expanded image of subtiiisin-untreated HIV-1lav-i preparation shows four spots (spots 7,8,14, and 25). Spot 25 completely disappeared on subtilisin treatment. This result suggests that CyP Ag gg exists on the viral surface. Spots 7,8, 14, and 25 were not detected in the image of the microvesicles. The spot numbers are correspond to those in Fig. 3. (B) Spots 7,8, and 25 on the...

Quantification by Two-Dimensional Gel Electrophoresis 2-DE is still considered the gold standard for quantitative proteomics [57,67]. This procedure relies on differential expression of proteins in control and test samples. Proteins from the two samples are separated by the 2-DE protocol, followed by staining with a suitable dye (e.g., CCB or SYPRO Ruby). Next, images of the stained spots are acquired, and spot densities are measured and compared by image analysis software to provide differential expression of proteins. Gel-to-gel variations can affect the precision of quantification by this procedme. 

---