Schistosoma japonicum GST Baculovirus-Insect cells Overexpression Lysate

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Schistosoma japonicum GST Baculovirus-Insect cells Overexpression Lysate: Product Information

Product Description
This Schistosoma japonicum GST overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of GST protein (Cat: 11213-HNAB) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect cells
Species
Schistosoma japonicum
Sequence Information
A DNA sequence encoding the Glutathione S-transferase (P08515) (Met 1-Lys 218), with additional 11 amino acids at the C-terminus, was expressed and purified.
Molecule Mass
The recombinant Glutathione S-transferase (GST) consists of 229 amino acids and predicts a molecular mass of 26.9 kDa. The apparent molecular mass of GST is approximately 28 kDa in SDS-PAGE under reducing conditions.

Schistosoma japonicum GST Baculovirus-Insect cells Overexpression Lysate: 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.

GST Background Information

Genetic engineershave used glutathione S-transferase to create the GST gene fusion system. This system is used to purify and detect proteins of interest. In a GST gene fusion system, the GST sequence is incorporated into anexpression vectoralongside the gene sequence encoding the protein of interest. Induction of protein expression from the vector's promoter results in expression of a fusion protein: the protein of interest fused to the GST protein. This GST-fusion protein can then be purified from cells via its high affinity for glutathione. GST is commonly used to createfusion proteins. The tag has the size of 22amino acids(roughly 26 KDa), which, compared to other tags like themyc- or theFLAG-tag, is quite big. However, many commercially-available sources of GST-tagged plasmids include athrombindomain for cleavage of the GST tag during protein purification.
References
    • Douglas KT . et al., 1987, Adv Enzymol Relat Areas Mol Biol.?59: 103- 67.?
    • Beckett GJ. et al., 1993, ?Adv Clin Chem. 30: 281-380.
    • Wilce MC. et al., 1994, ?Biochim Biophys Acta. 1205?(1): 1-18.
    • Leaver MJ. et al., 1998, ?Marine Environmental Research. 46?(1-5): 71-4.
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