Human PAPP-A2 HEK293 Overexpression Lysate 产品信息
Product Description
This Human PAPP-A2 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of PAPP-A2 protein (Cat: 10528-H08H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the human PAPPA2 mature form (NP_064714.2) corresponding to amino acid (Ser 234-Cys 1396) was expressed， with a carboxy-terminal polyhistidine tag.
Molecule Mass
The secreted recombinant human PAPPA2 consists of 1174 amino acids with the predicted molecular mass of 131 kDa. As a result of glycosylation, rhPAPPA2 migrates as an approximately 170-180 kDa band in SDS-PAGE under reducing conditions.
Human PAPP-A2 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 PAPP-A2 HEK293 Overexpression Lysate Alternative Names
Human PAPP-A2 Overexpression Lysate;Human PAPP-E Overexpression Lysate;Human PAPPE Overexpression Lysate;Human PLAC3 Overexpression Lysate
PAPP-A2 Background Information
Pappalysin-2/PAPP-A2 is the second member of the pappalysin family of metzincin superfamily, of which PAPP-A is the first member. There is no homology between the prepro-peptides of PAPP-A and PAPP-A2, but 46% of the residues of mature PAPP-A are also present in mature PAPP-A2. PAPP-A specifically cleaves insulin-like growth factor-binding protein(IGFBP)-4, one of six known modulators of IGF-I and –II, whereas PAPP-A2 specifically cleaved IGFBP-5 at one site, between Ser-143 and Lys-144. In contrast to the cleavage of IGFBP-4 by PAPP-A that strictly requires the presence of IGF, the cleavage of IGFBP-5 by PAPP-A2 was IGF-independent. Recent data firmly establish PAPP-A and IGFBP-4 as an important functional pair in several systems. Because of its close relationship with PAPP-A, both structurally and functionally, PAPP-A2 is a likely candidate for IGFBP-5 proteinase in many tissues and conditioned media where IGFBP-5 proteolysis has been reported.
References
Lisbeth S Laursen. et al., 2002, Vol. 277 (49): 47225-34. Michael T Overgaard. et al., 2001, The Journal of Biological Chemistry. 276 (24): 21849-53.
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