Experiments

November 2006
In this experiment, soluble oyster gill proteins were fractionated by ion exchange chromatography on a Waters High Performance Liquid Chromatography (HPLC) system, using a Waters PEEK Q anion exchange (AEX) column. A linear gradient of 0 to 0.7 M NaCl in 50 mM Tris pH 7.5 was applied over 6 minutes at 1 ml/min and fractions were collected. After evaluating the HPLC trace, the fractions were divided into four groups based on natural breaks in the absorbance profile, desalted using Ultra4 centrifugal concentrators, and diluted 5:1 in Guild BioSciences 2DE buffer. For each group, 2.5 ug of protein was fractionated by minigel 2DE using 3-10 pH range IPG strips and 10% total acrylamide SDS PAGE gels. All gels were silver stained. (Note: Only 2.5 ug of protein was applied because one of the AEX groups had a low protein concentration and equivalent quantities of protein were required for each gel.)

A 2DE gel of unfractionated soluble proteins is also included (below) for comparison.

The gels display that each AEX fraction contained a distinct set of proteins, demonstrating the ability of this technique to enrich for lower abundance proteins and increase overall resolution possible by 2DE.

November 2005
There are numerous ways to fractionate protein mixtures prior to proteomic analysis. In this experiment, soluble proteins derived from Saccharomyces cerevisiae were heat-treated for 5 minutes at 60 o C, placed on ice for 15 minutes, and centrifuged to pelletize the precipitated material. Using the method of Bradford, total protein was determined for the soluble heat-treated proteins and the pretreatment extract. The concentration of precipitated protein was calculated by subtracting the soluble heat-treated protein concentration from the initial protein concentration. Then, the precipitated protein was resolublized in Guild BioSciences 2DE buffer. Both the heat-stable and heat-labile protein fractions were analyzed by minigel 2DE, using a 3-10 pH range and 12% acrylamide, and silver stained.

These gels illustrate that the two protein maps have distinct protein sets, indicating that heat treatment can be used to enrich for either heat-stable or heat-labile proteins. Such effects could be used for purification of specific proteins or to increase the resolving power of 2DE through prefractionation. It is notable that when samples are resolublized under the denaturing conditions used in 2DE, protein precipitation is not a barrier. However, attempting to resolubilize precipitated proteins under non-denaturing conditions would be ineffective.

October 2005
Centrifugal ultrafiltration devices are commonly used for buffer exchange and concentration of protein solutions. From a methodological standpoint, it is useful to know whether the process alters proportions of the different proteins in the mixture via nonspecific interactions with the membrane or sides of the device. To explore this possibility, 1.8 ml of Vibrio fischeri soluble proteins (282 ug/ml protein) were applied to an Ultra4 centrifugal ultrafiltration unit and processed at 4000xG until a volume of 250 ul remained in the retenate. Then, 3.75 ml of 50 mM Tris pH 7.5 were added to the protein mixture, which was then centrifuged again at 4000xG until 190 ul of retenate remained. Total protein was measured by the method of Bradford and aliquots of soluble extract, both pre and post concentration, were diluted 5:1 in Guild BioSciences 2DE buffer. Five micrograms of each solution underwent analysis by minigel 2DE using a pH range of 4-7 and 12% total acrylamide, followed by silver staining.

The gels above show that the protein maps of the pre and post concentration extracts are very nearly identical. The post concentration extract appears to show increased resolution in the acidic high molecular weight region (enlarged in the Phoretix 2D images below).

This experiment provides evidence that buffer exchange and concentration of complex protein mixtures by centrifugal ultrafiltration devices, at least at similar protein concentrations, does not result in appreciable changes in 2DE protein map characteristics. It again demonstrates how 2DE can be used to evaluate protein preparative methods.

September-October 2005
In this experiment, a soluble protein extract was isolated from the luminescent bacteria Vibrio fischeri.

July 2004
In this experiment, a rapid two-step chromatographic prefractionation technique is used to quickly divide a complex protein mixture extracted from human HT-29 cell cultures into three distinct protein populations. This technique is key in Guild BioSciences’ high-resolution 2DE offering.

After desalting, fractions were analyzed by standard format 2DE using a 3-10 pH range, 12% acrylamide SDS PAGE gels, and silver staining. We used the 3-10 pH range so that the three fractions could be compared effectively with the unfractionated cytosol 2DE gel. However, increased resolution can be obtained using a 4-7 pH range for 2DE of fractions 2 and 3, while using a 4-7 pH range for 2DE of fraction 1. In comparison to the unfractionated cytosol 2DE gel, it is clear that many additional spots are visualized with the different fractions and that overall resolution is dramatically increased using sequential prefractionation.

November 2003
In this experiment, 1.3 ml of clarified soluble protein extract was isolated from oyster gill tissue. Using a molecular weight cutoff of 100 kd, the isolate was processed through a Centricon YM-100 centrifugal ultrafiltration device for 3.75 hr at 2000xG. Total protein determination was achieved using the Bradford total protein assay and the filtrate and retenate were diluted 5:1 in Guild BioSciences 2DE buffer. A 40 ug sample of protein was then analyzed by standard format 2DE using a pH range of 4-7 and 12% acrylamide. Gels were silver stained.

These gels display how 2DE can be used to assess and troubleshoot protein preparation. Specifically, the results show that size separation of a complex protein mixture by ultrafiltration can be ineffective. Presumably, size excluded proteins form a permeability barrier that prevents smaller proteins from passing through the membrane, disrupting the separation.