Isolation and purification of two major serum amyloid A isotypes SAA1 and SAA2 from the acute phase plasma of mice.
A new procedure was developed for isolation of two major serum amyloid A (SAA) isotypes SAA1 and SAA2 from acute-phase plasma of mice. The procedure included preparation of high-density lipoproteins (HDLs) and their separation by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The SAA proteins (Mr 12,000) were electroeluted and afterwards purified from SDS by gel permeation chromatography on a Fractogel TSK-40F column in aqueous 50% acetonitrile-0.1% TFA. Finally, the SAA proteins free from SDS were fractionated by high-performance liquid chromatography on a Vydac 214TP54 column (250 x 4.6 mm I.D., particle size 5 microm), yielding two major fractions with k=5.2 and k=5.5. The N- and C-terminal sequence analyses and mass spectrometry demonstrated the purity of these two major fractions and their identity with apo SAA1 (k=5.2) and apo SAA2 (k=5.5). The developed procedure is applicable to small amounts of pooled murine plasma (6-7 ml) and could be readily modified from small to large scale preparations.
Kaplan
B
B
Heller Institute of Medical Research, Sheba Medical Center, Tel-Hashomer, Israel.
Yakar
S
S
Balta
Y
Y
Pras
M
M
Martin
B
B
D000210:Acute-Phase Reaction / Q000097:blood*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D005591:Chemical Fractionation; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008075:Lipoproteins, HDL / Q000097:blood; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D017421:Sequence Analysis; D000685:Serum Amyloid A Protein / Q000302:isolation & purification*
D008075:Lipoproteins, HDL; D010446:Peptide Fragments; D000685:Serum Amyloid A Protein