Gel for Isoelectric Focusing
a gel and isoelectric technology, applied in the field of electrotrophoresis, can solve the problems of inability to use non-linear gradients for focusing peptides, uneven distribution of focused proteins/peptides in the gel, etc., to achieve the effect of reducing the diffusion of peptides, preventing depletion of denaturants, and high resolution
Inactive Publication Date: 2008-11-27
GE HEALTHCARE BIO SCI CORP
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Benefits of technology
[0010]The present invention provides an optimal immobilised pH gradient in IEF gels for peptide separation resulting in even distribution and high resolution of peptides in the gel in which the separation has been done. Each IEF-gel comprises a non-linear step-wise pH-gradient arranged in a continuous gel.
[0019]In a second aspect, the invention provides a kit comprising a dried or pre-cast IEF gel and a sample bridge comprising urea or other denaturant. The sample bridge is provided between one end of the gel and one of the electrodes, i.e. cathode or anode, and is used for loading of sample onto the gel. The sample bridge could be made of any suitable material, such as conventional paper bridge or hydrogel gel, such as acrylamide or agarose. The sample bridge is preferably saturated a high denaturant concentration, such as with 8M urea, and is provided on the anodic side of the IEF gel during focusing. The type of denaturant is dependent on the type of denaturant used in the gel. This sample bridge prevents depletion of denaturant from the IEF strip.
Problems solved by technology
A common problem with isoelectric focusing of proteins and / or peptides is that the focused proteins / peptides are unevenly distributed in the gel with bad resolution.
This non-linear gradient cannot be used for focusing of peptides.
Method used
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[0023]As shown in FIG. 1 human tryptic peptides originating from human proteins are unevenly along the x-axis representing their pI value.
[0024]According to the present invention, an uneven pH-gradient, see FIG. 2, should be immobilised in an IEF gel or strip which will result in an even distribution of tryptic peptides after isoelectric focusing. In FIG. 3 it is shown how to produce such an uneven pH-gradient.
[0025]The gels according to the invention are cast to a thickness of approximately 0.5 mm.
[0026]The gels may be cast to achieve the three separate pH-intervals in one gel. Alternatively, the gel may be cast as three separate gel portions, each representing the separate pH-interval, which thereafter are joined to form a continuous gel. The separate pieces could for example be joined with polyacrylamide bridges.
[0027]For better resolution of peptides the gel is optionally dried and re-swollen to less than 0.5 mm thickness. In a preferred embodiment, the gel is cast to a volume o...
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Abstract
The present invention relates to the field of electrophoresis and more specifically to a gel or strip for separating peptide components by isoelectric focusing by producing IPG (immobilised pH gradient) gels or strips in a novel way before focusing. More closely, the peptides are focused in a novel IPG gel including an uneven or non-linear pH gradient having at least three separate stepwise arranged pH-intervals. After focusing the peptide resolution is high and the peptides are evenly distributed along the gel.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a filing under 35 U.S.C. § 371 and claims priority to international patent application number PCT / SE2006 / 001449 filed Dec. 19, 2006, published on Jun. 28, 2007, as WO 2007 / 073293, which claims priority to patent application number 0502911-1 filed in Sweden on Dec. 22, 2005.FIELD OF THE INVENTION[0002]The present invention relates to the field of electrophoresis and more specifically to a gel or strip for separating peptide components by isoelectric focusing by producing IPG (immobilised pH gradient) gels or strips in a novel way. More closely, the peptides are focused in a novel IPG gel comprising an uneven or non-linear pH gradient comprising at least three separate pH-intervals.BACKGROUND OF THE INVENTION[0003]The isolation and separation of biomolecules, such as proteins and peptides, has become of an increased interest during the past years. Some biomolecules need to be isolated as a last step of a biotechnological...
Claims
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Login to View More IPC IPC(8): C07K1/28
CPCC07K1/28G01N27/44795G01N27/44747
Inventor BJELLQVIST, BENGTUHLEN, KRISTINA
Owner GE HEALTHCARE BIO SCI CORP