HA (monoclonal, Clone HA,C5)
AS18 4176 | Clonality: Monoclonal | Host: Mouse | Reactivity: Human influenza hemagglutinin (HA) tagged proteins
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Product Information
Immunogen
KLH-conjugated synthetic peptide: YPYDVPDYA
Host
Mouse
Clonality
Monoclonal
Purity
Immunogen affinity purified in PBS, pH 7.4,. Contains 0.05% sodium azide.
Format
Liquid
Quantity
100 ĩg
Storage
Store at -20°C, Store in small aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
Tested applications
Immunoprecipitation (IP), Immunolocalization (IL), Western Blot (WB)
Recommended dilution
1:200 (IP, IL), 1:1000 (WB)
Reactivity
Confirmed reactivity
HA-tagged proteins in transfected mammalian cells or expressed under native promoter
Application examples
Application examples
Application example
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
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
Additional information
Background
Background
HA-tag is derived from a human influenza hemagglutinin HA-molecule corresponding to amino acids 96-106 and is used as a general epitope tag in expression vectors.
Product citations
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