Human BTD HEK293 Overexpression Lysate 产品信息
Product Description
This Human BTD overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of BTD protein (Cat: 15661-H08H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the human BTD (P43251) (Met1-Asp543) with a C-terminal polyhistidine tag was expressed.
Molecule Mass
The recombinant human BTD comprises 513 amino acids and has a predicted molecular mass of 58.2 kDa. The apparent molecular mass of the protein is approximately 66-76 kDa in SDS-PAGE under reducing conditions.
Human BTD HEK293 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.
Human BTD HEK293 Overexpression Lysate Alternative Names
Human BTD Overexpression Lysate
BTD Background Information
Biotinidase, also known as biotinase and BTD, is a ubiquitous mammalian cell enzyme which expressed at high levels in the liver, serum, and kidney. Its primary function is to cleave biotin from biocytin, preserving the pool of biotin for use as a cofactor for biotin dependent enzymes, namely the 4 human carboxylases. Biotinidase also recycles biotin from enzymes in the body that use it as a helper component in order to function. These enzymes, known ascarboxylases, are important in the processing of fats, carbohydrates, and proteins. Biotin is attached to these carboxylase enzymes through an amino acid (the building material of proteins) called lysine, forming a complex calledbiocytin.
References
Cole H., et al.,(1994), Human serum biotinidase. cDNA cloning, sequence, and characterization. J. Biol. Chem. 269:6566-6570. Knight H.C., et al., (1998), Structure of the human biotinidase gene.Mamm. Genome 9:327-330. Ota T., et al.,(2004), Complete sequencing and characterization of 21,243 full-length human cDNAs.Nat. Genet. 36:40-45.
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