Convenient high-resolution isoelectric focusing (IEF) method for the separation of alpha1-proteinase inhibitor (A1PI) isoforms in A1PI concentrates.
Currently, high-resolution separation of A1PI is done in highly specialized laboratories using gels made in-house. This paper presents a high-resolution method for the separation of A1PI concentrates and human plasma using commercially available gels. Hybrid IEF was performed with carrier ampholytes and the gels were stained with Coomassie Brilliant Blue G-250. In addition, a sensitive immunoblotting procedure is described. The IEF method allowed the reproducible and convenient determination of the IEF pattern of A1PI in concentrates including resolution of glycan-dependent isoforms and isoproteins with secondary modifications such a C-terminal Lys-truncation. Furthermore, a shift in the IEF pattern of A1PI occurring upon reduction could be detected. Finally, in combination with a sample pretreatment step, the method proved able to monitor complex A1PI isoform patterns in samples with low A1PI concentrations as present for example in bronchoalveolar lavage solutions.
Weber
Alfred
A
Baxter BioScience, Industriestrasse 67, A-1220 Vienna, Austria.
Engelmaier
Andrea
A
Baxter BioScience, Industriestrasse 67, A-1220 Vienna, Austria.
Owen
Maurice C
MC
Canterbury Scientific Limited, 14 Pope Street, Christchurch 8011, New Zealand.
Schwarz
Hans-Peter
HP
Baxter BioScience, Industriestrasse 67, A-1220 Vienna, Austria.
Turecek
Peter L
PL
Baxter BioScience, Industriestrasse 67, A-1220 Vienna, Austria. Electronic address: peter_turecek@baxter.com.
D015153:Blotting, Western; D005782:Gels; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods; D020033:Protein Isoforms / Q000097:blood; D000515:alpha 1-Antitrypsin / Q000097:blood*
D005782:Gels; D020033:Protein Isoforms; D000515:alpha 1-Antitrypsin