Guidelines To Generate Overlaps Between 2-D Gels and Blots

Two-dimensional (2-D) electrophoresis is a widely used gel-based method for separating protein mixtures by charge (isoelectric point) and size (molecular weight). After 2-D electrophoresis (2-DE; described in detail in the Bio-Rad Applications & Technologies section), gels can be processed for protein detection via total protein staining and/or western blotting. Western blotting analysis following 2-DE separation is particularly useful in at least two important applications: (1) identification of pathogen-derived proteins that are highly immunogenic (have potential as vaccine candidates), and (2) evaluation of the range of host cell protein (HCP) impurities detectable by polyclonal antibodies used to monitor HCP impurities in purified biopharmaceuticals. For both applications, it is critical to generate a match rate from an overlay of the 2-DE and western blotting analyses. Bio-Rad’s PDQuest 2-D analysis software can be used to easily generate match rates. We describe here, step-by-step guidelines for generating this information using PDQuest software.

Guidelines for Match Rate Generation

Capture images using Bio-Rad’s ChemiDoc™ MP imaging system. Generate images using exposure times that give good image quality without saturation. For imaging the blot, create a custom protocol for chemiluminescence using 1 x 1 binning. Use the same zoom settings for imaging both the fluorescent stain and the chemiluminescent western blot.

Use Image Lab™ software to crop both images to the edge of the membrane. If necessary, first rotate each image so that the edges of the membrane are parallel with the frame of the image. This will ensure that the stained blot image and the western blot image are in register for PDQuest software analysis. Export the images for analysis (File>Export>Export for Analysis).

Launch PDQuest software. Open both of the files exported from Image Lab software.

Step-by-step guidelines for using the PDQuest software to generate match rates.

Fig. 1A. Crop the images to include only the area containing well-resolved protein spots. Use the Advanced Crop function to ensure that the cropped images are registered with each other. Place the crosshairs on a recognizable feature common to both images (Edit > Image > Advanced Crop > Define Crop Area).
Fig. 1B. Crop the images to include only the area containing well-resolved protein spots. Use the Advanced Crop function to ensure that the cropped images are registered with each other. Place the crosshairs on a recognizable feature common to both images (Edit > Image > Advanced Crop > Define Crop Area).
Fig. 2. Create a new experiment (File > New Experiment). Select both of the cropped images. Move through the next set of screens by clicking Next. This will select the default options. Follow the instructions on the Spot Detection Parameter Wizard. Increase the sensitivity to maximize the number of spots identified.
Fig. 3. Select Proceed. In the next dialog box, select the cropped image of the stained gel as the master gel. Select Add unmatched spots from all gel images to the Master gel. This will create an artificial image containing all of the detected spots from both the stained blot and the immunodetected western blot.
Fig. 4. Choose default values by clicking Next through the successive dialog boxes. The software will calculate matches and display the master image, the blot images, and an Experiment Summary with match rates.
Fig. 5. Match Rate 2 for the western blot image is the ratio of the number of protein spots recognized by the antibody to the total number of proteins detectable in the sample.
Fig. 6. Choosing View > Spot > Show Crosshairs or pressing F5 will show the detected spots.
Fig. 7. To show matched spots, select Analyze>Analysis Set Manager,… then Create and Matching.
Fig. 8. The resulting images will have identified spots marked with a colored “+” and matched spots marked with an “x” in a different color. This will aid in determining whether or not a spot identification or match is valid.
Fig. 9. A more accurate match rate can be determined by deleting incorrectly identified spots. These can often be visually identified near the edges of the image. In this case, a series of spots along the top edge of the blot image probably do not correspond to real proteins. Spurious spots are deleted by selecting Edit > Spot > Remove Spot. The spots are erased by clicking on the spot or drawing a box around a set of spots. This operation should be performed both on the raw 2-D image and on the master.
Fig. 10. Selecting Edit>Experiment Summary will display the corrected match rate.
Fig. 11. If desired, the match may be further refined by manually adding and deleting spots. Spot matches may be systematically verified using the Spot Review Tool (Analyze > Spot Review).