Anti-SNAP/Clip Tag

Samples: From the left: Arabidopsis thaliana wildetype Arabidopsis thaliana expressing pUBQ:SNAP-TUA5

10 days old Arabidposis thaliana plants were grown in vitro and 100 mg of frozen tissue was grind in 300 ul Laemmli buffer (125 mM Tris HCl pH 6.8, 2.5 % SDS, 20% (v/v) glycerol), 0.01% bromophenol blue, +20 mg/ml DTT) was denatured at 65°C for 10 minutes and subjected to SDS-PAGE gel electrophoresis with 15 µg of protein load/line, at room temperature followed by semi dry transfer at room temperature to PVDF membrane (pore size 0.2 µm). Blocking was done with 5% milk 0.02% Tween20 in TBS for 1h at RT with agitation. Primary antibody in 2.5% milk, 0.02% Tween20 in TBS were used at 1:2000/ON at 4°C with agitation. Washing was done three times for 15 minutes in 2.5% milk, 0.02% Tween20 in TBS, followed by incubation with the secondary antibody in 2.5% milk, 0.02% tw20 in TBS diluted to 1:20 000/2h at RT with agitation, followed by three times wash for 15 min in 2.5% milk, 0.02% tw20 in TBS. Last wash was performed only in TBS. Antibody binding was visualized with West Dura (Thermofisher, ref 34075). Exposure time was 1 second.

To decrease background signal, this antibody can be used in higher dilution 1: 5000/1h RT incubation combined with less sensitive detection reagent.

Courtesy of Dr.Alexandre Martinière, Institut for Plant Sciences of Montpellier (IPSiM), France