Plant urease detection with commercial antibodies

 

Plant urease detection with commercial antibodies


Antibodies from three different suppliers were tested. Results were very similar, irrespective of source.
 
 
Antibody Supplier Host animal Purity Storage buffer
Polyclonal anti-jackbean seed urease antibody Europa Bioproducts Ltd, Cambridge Rabbit IgG fraction PBS+0.1% sodium azide
Polyclonal anti-jackbean seed urease antibody Rockland, Gilbertsville, Pennsylvania Rabbit serum PBS+0.01% sodium azide
Polyclonal anti-jackbean seed urease antibody Biomedia, Foster City, California Goat affinity purified 50 mM borate, pH 8.0; 150 mM NaCl; 0.05% sodium azide

 

Results from experiments with potato extracts



 


A: Western blot of protein extracts from different organs of glasshouse-grown potato plants (cv. Désirée) at the flowering stage. The first lane contains purified jackbean urease (Boehringer). The arrow indicates the position of urease.
Extracts were prepared with the large scale extraction protocol, and 70 microliter per lane was loaded on a 10% SDS gel (thickness 1.5 mm). The blot was performed in Towbin buffer + 0.02% SDS for 2 h 30 min in a BioRad tank blot apparatus.
Membrane blocking: 2% dry milk; 2% BSA in PBS; anti-urease antibody (Europa): 1:2000 diluted in blocking solution, over-night at 4°C. Washes: 3 x 5 min PBS + 1 M NaCl + 0.2% Tween 20. Secondary antibody: anti rabbit IgG alkaline phosphatase conjugate (1:5000; Sigma).
B: Western blot of protein extracts from different organs of field-grown just emerged potato plants. The last lane contains the positive control. The arrow indicates the position of urease. Extracts were obtained with the large scale protocol, 80 microliter per lane was loaded. Experimental conditions were very similar to A, except that washes of the blot were performed with PBS + 0.2% Tween 20 (no NaCl).
 

Results from experiments with (leaf) extracts from several plants

 

 


 


Urease detection on a Western blot containing (leaf) protein extracts from different plants. Leaf extracts were obtained with the small scale extraction procedure. Jackbean seed extract was obtained from jackbean seed meal. Equal volumes were loaded per lane. The extract in lane e (potato,60°C) was incubated in a 60°C water bath for 5 min and precipitates were removed before loading (urease activity is stable at 60°C, see Heat stability of urease). Experimental conditions were similar as described above (Antibody: Europa 1:3000; washes with PBS + 0.4% Tween 20 + 200 mM NaCl).
 

Conclusions

Claus-Peter Witte, 2001
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