[Purification from goat antiserum of immunoglobulins against G6PD from rabbits].
DEAE Affi-Gel Blue (Bio-Rad) provides an efficient and rapid fractionation of human serum proteins by a single chromatographic step. When goat serum is applied to the matrix and chromatography is performed following the procedure utilized for the human serum proteins, the elution pattern changes and the Ig purification is not satisfactory. We achieved a better Ig purification from goat serum by the following improved procedure. We performed first an AS-40 fractionation followed by extensive dialysis in 50 mM Na-citrate pH 5.7. The sample was then loaded onto a P11 column equilibrated in the same buffer. The fraction eluted at Vo contained total IgG and the other serum proteins, except beta-globulins which were eluted with 0.24 M phosphate. Peak 1 concentrated and dialyzed in 20 mM phosphate buffer pH 8 was then applied to a DEAE Affi-Gel Blue column, equilibrated in the same buffer. Two protein peaks were eluted from this column and electrophoretically characterized as: peak 1, containing a pure Ig fraction (70% yield), peak 2 with albumin and other contaminating serum proteins. When goat antiserum is obtained against a specific protein, our technique may be suitably employed to purify polyclonal antibodies for immunoprecipitation studies.
Baronciani
L
L
Istituto di Chimica Biologica, Università di Urbino.
Rapa
S
S
Mannello
F
F
Benedetti
C
C
Ninfali
P
P
D000818:Animals; D002852:Chromatography, Ion Exchange; D005954:Glucosephosphate Dehydrogenase / Q000276:immunology*; D006041:Goats / Q000097:blood* / Q000276:immunology; D007106:Immune Sera; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification*; D011817:Rabbits; D013045:Species Specificity
D007106:Immune Sera; D007074:Immunoglobulin G; D005954:Glucosephosphate Dehydrogenase
Purificazione da antisiero caprino di immunoglobuline anti-G6PD di coniglio.