HA (monoclonal, Clone HA,C5)
AS18 4176 | Clonality: Monoclonal | Host: Mouse | Reactivity: Human influenza hemagglutinin (HA) tagged proteins
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Tagged protein is expressed under native promoter and so IP is performed prior to Western Blotting. Starting material of 200mg of root tissue was used for membrane protein extraction with buffer components Tris pH7,5, Tris pH 6.8, D-Sorbitol, casein, Na EDTA, Na EGTA, NaF, Na molybdate, β-glycerophosphate, KH2PO4 7.8 and Protease inhibitor cocktail and denatured with 1x Lameli buffer at 85°C for 10 min were separated on 10 % SDS-PAGE and blotted 1.5h to PVDF using wet transfer. Blot was blocked with 5% milk for 2h/RT with agitation. Blot was incubated in the primary antibody at a dilution of 1: 5 000 (both anti-GFP & anti-HA) for ON/4°C with agitation in TBS-T. The antibody solution was decanted and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in Agrisera matching secondary antibody (anti-mice HRP, anti-Chicken HRP) diluted to 1:10 000, 1:5000, in for 2h/RT with agitation. The blot was washed as above and developed with chemiluminescence detection reagent. Exposure time was 2mins (anti-GFP) and 2sec (anti-HA).
Courtesy of Stanislava Sládečková, Developmental and Cell Biology of Plants, Masaryk University, CEITEC, Czech Republic