Influenza A H5N2 (A/American green-winged teal/California/HKWF609/2007) Hemagglutinin (HA1 Subunit) HEK293 Cell Lysate (WB positive control)

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Influenza A H5N2 (A/American green-winged teal/California/HKWF609/2007) Hemagglutinin (HA1 Subunit) HEK293 Cell Lysate (WB positive control): Product Information

Product Description
This H5N2 Hemagglutinin / HA overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Hemagglutinin / HA protein (Cat: 11699-V08H1) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Species
H5N2
Sequence Information
A DNA sequence encoding the N-terminal segment (Met 1-Arg 342) of the influenza A hemagglutinin (A/American green-winged teal/California/HKWF609/2007(H5N2)) (ACF47563.1), termed as HA1, was fused with a C-terminal polyhistidine tag.
Molecule Mass
The secreted recombinant HA1 subunit of influenza A H5N2 HA (A/American green-winged teal/California/HKWF609/07 (H5N2)) comprises 337 amino acids and has a predicted molecular mass of 38.2 kDa. As a result of glycosylation, it migrates as an approximately 45-50 kDa band in SDS-PAGE under reducing conditions.

Influenza A H5N2 (A/American green-winged teal/California/HKWF609/2007) Hemagglutinin (HA1 Subunit) HEK293 Cell Lysate (WB positive control): Usage Guide

Preparation Method
Cell lysate was prepared by homogenization of the over-expressed cells in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.
Lysis Buffer
Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.
Recommend Usage
1.  Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2.  Re-dissolve the pellet using 200μL pure water and boil for 2-5 min.
Sample Buffer
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
Store at 4℃ for up to twelve months from date of receipt. After re-dissolution, aliquot and store at -80℃ for up to twelve months. Avoid repeated freeze-thaw cycles.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.

Hemagglutinin / HA Background Information

The influenza viral Hemagglutinin (HA) protein is a homo trimer with a receptor binding pocket on the globular head of each monomer.HA has at least 18 different antigens. These subtypes are named H1 through H18.HA has two functions. Firstly, it allows the recognition of target vertebrate cells, accomplished through the binding to these cells' sialic acid-containing receptors. Secondly, once bound it facilitates the entry of the viral genome into the target cells by causing the fusion of host endosomal membrane with the viral membrane.The influenza virus Hemagglutinin (HA) protein is translated in cells as a single protein, HA, or hemagglutinin precursor protein. For viral activation, hemagglutinin precursor protein (HA) must be cleaved by a trypsin-like serine endoprotease at a specific site, normally coded for by a single basic amino acid (usually arginine) between the HA1 and HA2 domains of the protein. After cleavage, the two disulfide-bonded protein domains produce the mature form of the protein subunits as a prerequisite for the conformational change necessary for fusion and hence viral infectivity.
Full Name
Harvey rat sarcoma viral oncogene homolog
References
  • White JM, Hoffman LR, Arevalo JH, et al. (1997). ""Attachment and entry of influenza virus into host cells. Pivotal roles of hemagglutinin"". In Chiu W, Burnett RM, Garcea RL. Structural Biology of Viruses.
  • Suzuki Y (March 2005). ""Sialobiology of influenza: molecular mechanism of host range variation of influenza viruses"". Biol. Pharm. Bull. 28 (3): 399–408.
  • Senne DA, Panigrahy B, Kawaoka Y, et al. (1996). ""Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential"". Avian Dis. 40 (2): 425–37

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