Product Description
This Human Thrombopoietin overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Thrombopoietin protein (Cat: 10381-H07H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the mature form of human THPO (NP_000451.1) (Ser 22-Gly 353) was fused with a polyhistidine tag at the N-terminus.
Molecule Mass
The recombinant human THPO consists of 348 amino acids with the predicted molecular mass of 37.6 kDa. As a result of glycosylation, rhTHPO migrates as an approximately 80 kDa band in SDS-PAGE under reducing conditions.
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 MGDF Overexpression Lysate;Human MKCSF Overexpression Lysate;Human ML Overexpression Lysate;Human MPLLG Overexpression Lysate;Human THCYT1 Overexpression Lysate;Human TPO Overexpression Lysate
Thrombopoietin (TPO or THPO), also known as myeloproliferative leukemia virus ligand (c-Mpl), is a hematopoietic growth factor belonging to the EPO/TPO family. The thrombopoietin protein is produced mainly by the liver and the kidney that regulates the production of platelets by the bone marrow. Thrombopoietin protein stimulates both proliferation of progenitor megakaryocytes and their maturation to platelet-producing megakaryocytes, and also accelerates the recovery of platelets. Thrombopoietin protein is involved in cardiovascular disease as it regulates megakaryocyte development and enhances platelet adhesion/aggregation. It has been identified that surface c-MPL, the receptor for thrombopoietin protein, binds to the ligand and mediates the action.
References
Ryu T,et al.(2003) Thrombopoietin-producing hepatocellular carcinoma. Intern Med. 42(8): 730-4. Higashihara M,et al. (2003) Thrombopoietin-producing tumor. Intern Med. 42(8): 632-3? 
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