Human Ribonuclease A HEK293 Overexpression Lysate

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Human Ribonuclease A HEK293 Overexpression Lysate: 产品信息

产品描述
This Human Ribonuclease A overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Ribonuclease A protein (Cat: 13468-H08H) from the overexpression lysate was verified.
表达宿主
HEK293 Cells
种属
Human
蛋白构建信息
A DNA sequence encoding the human RNASE1 (P07998) (Met1-Thr156) was expressed with a polyhistidine tag at the C-terminus.
分子量
The recombinant human RNASE1 comprises 139 amino acids and has a predicted molecular mass of 16 kDa. The apparent molecular mass of the protein is approximately 29 ,26 and 22 kDa in SDS-PAGE under reducing conditions

Human Ribonuclease A HEK293 Overexpression Lysate: 使用指南

制备方法
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.
裂解缓冲液
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.
使用建议
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.
缓冲液
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
稳定性 & 储存条件
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.
应用
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.

Human Ribonuclease A HEK293 Overexpression Lysate: 别称

Human RAC1 Overexpression Lysate; Human RIB1 Overexpression Lysate; Human RNS1 Overexpression Lysate

Ribonuclease A 背景信息

RNase A, also known as ribonuclease A and RNASE1, belongs to ribonuclease A superfamily. It is a pancreatic-type of secretory ribonuclease. RNase A is a basic protein and its many positive charges are consistent with its binding to RNA (a poly-anion). More generally, RNase A is unusually polar or, rather, unusually lacking in hydrophobic groups, especially aliphatic ones. As an endonuclease, RNase A cleaves internal phosphodiester RNA bonds on the 3'-side of pyrimidine bases. It prefers poly(C) as a substrate and hydrolyzes 2',3'-cyclic nucleotides, with a pH optimum near 8.0. RNase A is monomeric and more commonly acts to degrade ds-RNA over ss-RNA. Alternative splicing occurs at this locus and four transcript variants encoding the same protein have been identified.
全称
ribonuclease, RNase A family, 1 (pancreatic)
参考文献
  • Tubert P, et al. (2011) Interactions crucial for three-dimensional domain swapping in the HP-RNase variant PM8. Biophys J. 101(2):459-67.
  • Vinayagam A, et al. (2011) A directed protein interaction network for investigating intracellular signal transduction. Sci Signal. 4(189):rs8.
  • Fischer S, et al. (2011) Expression and localisation of vascular ribonucleases in endothelial cells. Thromb Haemost. 105(2):345-55.
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