This project consists of an effort to obtain the alkaline two-dimensional map of human resting neutrophil proteins. Being able to fuse the acidic map (already completed in our laboratory) with the current project might be important to observing the expression of a large number of proteins from the whole cell extract which, in turn, is essential for future research in, for instance, anti-inflammatory drug development or diagnostic molecular markers.
Proteins with pI higher than 6 are normally poorly represented with standard isoelectric focalization (IEF) techniques and regular rehydration solutions due to high protein hydrophobicity, protein precipitation, oligomerization and problems in buffering, which demonstrates the necessity of protocol optimization for this kind of procedure.
In order to improve spot resolution in the gel, many new features were introduced into the protocol based on literature-available protocols. The experiments were conducted with Lysis Buffer (Urea 7M, Thiourea 2M, Triton X-100 1%v/v, Pharmalyte 0.5%v/v, DTT 65mM e Isopropanol 10%v/v) for sample dilution. The IEF occurred on 18cm pH6-11 commercial IPG strips and both IPG-phor and Multiphor-II were tested. The second dimension was performed in BioRad II electrophoresis cubes filled with pH 8.3 Tris-Glycine/SDS buffer. Modified Ammonium Silver Staining and Regular Silver Satining were the staining protocols used on the gels.
Among the many protocol additions tested in the experiments were: the use of Isopropanol 10% in the sample preparation and Lysis Buffer; use of constant entrance of migrating reducing agents, such as DTT (this is one of the features that requires the usage of Multiphor-II instead of IPG-phor); application of organic disulfides for streak elimination; enhanced IEF time, which might include longer runs to avoid protein precipitation or shorter runs with higher voltages; voltage application during part of the rehydration step; use of Glycerol in the rehydration solution; and in-gel rehydration. Those changes, associated or not, allowed us to obtain good separation profiles that include low presence of vertical and horizontal streaking, relatively clear background and good spot geometry and figures of up to 350 spots distributed in a wide mass and pH range.