Identification of Acanthamoeba ∞ tubulin by immunoblotting

Cell Biology

International

Identification

Reports,

of Acanthamoeba

T M Preston

and E P Urquhart

Department

of

zoology)

Vol. 9, No. 3, March

ectubulin

1985

283

by Immunoblotting

University

College

London,

London

wclE ~BT network of fluorescence microscopy an extensive using BY been demonstrated in microtubules has recently cytoplasmic Acanthamoeba (1). A substratum-attached cytoskeletons of monoclonal antibody (YOL l/34) to yeast tubulin (2) was used for as the primary antibody in this study. Here we report for the first time an apparent molecular weight for Acanthamoeba tubulin identified by Western blotting with this same antibody 15Opl after SDS gel electrophoresis of amoeba1 cytoskeletons. of detergent stock (2% nonidet P40, 60mM PIPES, 25mM HEPES, 1OmM EGTA, 4mM MgC12, 2mM benzamidine-hydrochloride, 0.2mM phenylmethylsulphonyl fluoride, 20ugml-1 leupeptin, 40ugml-1 soyabean trypsin inhibitor, pH 6.9) was added to 150~1 of freshly harvested & washed amoebae in suspension (lo7 cells ml-l in 5OmM NaCl). Extraction proceeded for 10' on ice followed by 10' at 22°C. Cytoskeletons were then sedimented (4' at 11,600xg) and the supernatant discarded. The pellet was washed by addition of 300~1 of detergent stock diluted 1:l with sterile 50mM NaCl. After further the 1 centrifugation tubes containing the pellets were inverted and 1 2 allowed to dry. These cytoskeleton preparations were solubilized in 2.3% SDS in sample buffer and run on 7.5% acrylamide SDS-tris-glycine slab FIGURE 1. gels. Following Western blotting, binding of YOL l/34 to the nitrocellulose transfer was visualized by a second layer immunoperoxidase reaction. Our results (fig 1) show that YOL l/34 binds to a single polypeptide band (fig 1, track 1) that migrates, under the conditions used, at a speed intermediate to that of the oc(fig 1, track 2) and p subunits of bovine brain tubulin. A comparison with molecular weight standards gives an apparent molecular weight for this Acanthamoeba dtubulin band of 55kD. (1) (2)

Preston Kilmartin

TM (1984) ms submitted JV et al (1982) J cell

Biol

93:576-582

Acknowledgements Drs T Butt, JS Hyams 6 CA King

Received

:

5th December 1984

0309-1651/85/030283-01/$03.00!0

Accepted:

3rd January

0 1985 Academic

1985

Press Inc. (London)

Ltd.