Human NAMPT Baculovirus-Insect cells Overexpression Lysate

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Human NAMPT Baculovirus-Insect cells Overexpression Lysate: Product Information

Product Description
This Human NAMPT overexpression lysate was created in Baculovirus-Insect cells and intented for use as a Western blot (WB) positive control. Purification of NAMPT protein (Cat: 10990-H20B) from the overexpression lysate was verified.
Expression Host
Baculovirus-Insect cells
Species
Human
Sequence Information
A DNA sequence encoding the human NAMPT (P43490) (Met 1-His 491 was fused with the N-terminal polyhistidine-tagged GST tag at the N-terminus.
Molecule Mass
The recombinant human NAMPT/GST chimera consists of 728 amino acids and has a calculated molecular mass of 83.3 kDa. It migrates as an approximately 75 kDa band in SDS-PAGE under reducing conditions.

Human NAMPT 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.

Human NAMPT Baculovirus-Insect cells Overexpression Lysate: Alternative Names

Human 1110035O14Rik Overexpression Lysate; Human PBEF Overexpression Lysate; Human PBEF1 Overexpression Lysate; Human VF Overexpression Lysate; Human VISFATIN Overexpression Lysate

NAMPT Background Information

Nicotinamide phosphoribosyltransferase (NAMPT), also known as pre-B-cell colony-enhancing factor 1 (PBEF1) or visfatin, is an enzyme belonging to the family of glycosyltransferases, to be specific, the pentosyltransferases. This enzyme participates in nicotinate and nicotinamide metabolism. This enzyme catalyzes the condensation of nicotinamide with 5- phosphoribosyl-1- pyrophosphate to yield nicotinamide mononucleotide, one step in the biosynthesis of nicotinamide adenine dinucleotide. NAMPT is also considered as an essential enzyme mediating granulocyte colony-stimulating factor (G-CSF)-triggered granulopoiesis in healthy individuals and in individuals with severe congenital neutropenia. Intracellular NAMPT and NAD+ amounts in myeloid cells, as well as plasma NAMPT and NAD+ levels, were increased by G-CSF treatment of both healthy volunteers and individuals with congenital neutropenia.
Full Name
nicotinamide phosphoribosyltransferase
References
  • Skokowa J, et al. (2009) NAMPT is essential for the G-CSF-induced myeloid differentiation via a NAD+-sirtuin-1-dependent pathway. Nat Med. 15(2): 151-8.
  • Samal B, et al. (1994) Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol Cell Biol. 14 (2): 1431-7.
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