Purification of protein from a crude mixture through SDS-PAGE transfer method.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer method was used for purification and enrichment of the protein from crude sample. Coomassie bluc/ZnSO4 stained protein band(s) containing intact polyacrylamide gel were loaded on to another polyacrylamide gel either alone or as pooled gel bands. Two/three bands were combined together and arranged tightly over one another, sealed with stacking gel and ran in another gel, which was quite useful for enrichment and purification of a particular protein from a complex mixture. Recovery of protein by gel transfer method was found to be 70% in case of ZnSO4 staining, whereas around 30% recovery was possible, following Coomassie blue staining. The method described here for purification of protein(s) from a complex mixture, following gel transfer procedure could be useful for further characterization of the desired protein.

Isolation of bovine plasma albumin by liquid chromatography and its polymerization for use in immunohematology

The aim of the method described here is to remove hemoglobin, the major contaminant in the bovine plasma obtained from slaughterhouses, by adding a mixture of 19 percent cold ethanol and 0.6 percent chloroform, followed by fibrinogen and globulin precipitation by the Cohn method and nonspecific hemagglutinin by thermocoagulation. The experimental volume of bovine plasma was 2,000 ml per batch. Final purification was performed by liquid chromatography using the ion-exchange gel DEAE-Sepharose FF. The bovine albumin thus obtained presented > or = 99 percent purity, a yield of 25.0 + or 1.2 g/l plasma and >71.5 percent recovery. N-acetyl-DL-tryptophan (0.04 mmol/g protein) and sodium caprylate (0.04 mmol/g protein) were used as stabilizers and the final concentration of albumin was adjusted to 22.0 percent (w/v), pH 7.2 to 7.3. Viral inactivation was performed by pasteurization for 10 h at 60 degres C. The bovine albumin for the hemagglutination tests used in immunohematology was submitted to chemical treatment with 0.06 percent (w/v) glutaraldehyde and 0.1 percent (w/v) formaldehyde at 37 degrees C for 12 h to obtain polymerization. A change in molecular distribution was observed after this treatment, with average contents of 56.0 percent monomers, 23.6 percent dimers, 12.2 percent trimers and 8.2 percent polymers. The tests performed demonstrated that this polymerized albumin enhances the agglutination of Rho(D)-positive red cells by anti-Rho(D) serum, permitting and improving visualization of the results