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The elastase inhibitors of swine serum: an improved method for the isolation of alpha 2-macroglobulin
Abstract:
A previous communication from this laboratory as well as one from another described the separation of alpha 2-macroglobulin from swine serum. The products from both laboratories contained, in addition to alpha 2-macroglobulin, an additional macroglobulin contaminant with alpha 2-globulin mobility. Due to their physicochemical similarity these macroglobulins are not resolved using conventional column procedures such as ion exchange chromatography and gel filtration. Subsequent experiments have shown that immunoelectrophoretically pure swine alpha 2-macroglobulin is present, in good yield (65%) in the breakthrough effluent of columns of Bio-Gel A-1.5m-Reactive Blue 2 while the contaminating macroglobulin is tightly bound. The production of highly purified swine alpha 2-macroglobulin utilizing this observation is the subject of the present report. The product of the separation was found to be homogeneous when subjected to immunoelectrophoresis, at a concentration of 14-16 mg/ml, and diffused against antiswine whole serum antibody. The production of monospecific antibody, a more stringent test for homogeneity, resulted when the purified alpha 2-macroglobulin was injected into rabbits. Physicochemical analyses on the purified product showed that swine and human alpha 2-macroglobulins are true homologs.
Insights
Researchers developed a new method to purify swine alpha 2-macroglobulin, a protein found in blood. This technique effectively separates it from contaminants, yielding a highly pure product for further study.
Area of Science:
- Biochemistry
- Immunology
- Proteomics
Background:
- Previous methods for isolating swine alpha 2-macroglobulin yielded products contaminated with other macroglobulins.
- Physicochemical similarities between alpha 2-macroglobulin and contaminants made separation difficult using conventional chromatography.
Purpose of the Study:
- To develop a highly efficient method for purifying swine alpha 2-macroglobulin.
- To confirm the homogeneity and physicochemical properties of the purified swine alpha 2-macroglobulin.
Main Methods:
- Affinity chromatography using Bio-Gel A-1.5m-Reactive Blue 2 was employed.
- Purified protein homogeneity was assessed via immunoelectrophoresis and monospecific antibody production.
Main Results:
- A novel method utilizing Bio-Gel A-1.5m-Reactive Blue 2 successfully separated swine alpha 2-macroglobulin from contaminants.
- High yields (65%) of immunoelectrophoretically pure swine alpha 2-macroglobulin were achieved.
- The purified protein demonstrated homogeneity and physicochemical similarity to human alpha 2-macroglobulin.
Conclusions:
- Affinity chromatography with Bio-Gel A-1.5m-Reactive Blue 2 is an effective technique for obtaining highly purified swine alpha 2-macroglobulin.
- Swine and human alpha 2-macroglobulins are confirmed as true homologs.
- This purification method provides a reliable source of pure swine alpha 2-macroglobulin for research.