This Human IgG1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of IgG1 protein (Cat: 10702-HNAH) from the overexpression lysate was verified.
A DNA sequence encoding the human IgG1 Fc region (P01857-1) (Glu99-Lys330) (one aa mutation, 103 Cys/Ser) was expressed.
The recombinant human IgG1 Fc consists of 232 amino acids and has a predicted molecular mass of 26 kDa. As a result of glycosylation, the apparent molecular mass of rhFc is approximately 32 kDa in SDS-PAGE under reducing conditions.
Human IgG1 HEK293 Overexpression Lysate: 用法
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.
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.
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
安定性 & 保存条件
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.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human IgG1 HEK293 Overexpression Lysate: 別名
Human IgG1 Fc Overexpression Lysate; Human Ighg1 Overexpression Lysate
As a monomeric immunoglobulin that is predominately involved in the secondary antibody response and the only isotype that can pass through the human placenta, Immunoglobulin G (IgG) is synthesized and secreted by plasma B cells, and constitutes 75% of serum immunoglobulins in humans. IgG antibodies protect the body against the pathogens by agglutination and immobilization, complement activation, toxin neutralization, as well as antibody-dependent cell-mediated cytotoxicity (ADCC). IgG tetramer contains two heavy chains (5 kDa ) and two light chains (25 kDa) linked by disulfide bonds, that is the two identical halves form the Y-like shape. IgG is digested by pepsin proteolysis into Fab fragment (antigen-binding fragment) and Fc fragment ("crystallizable" fragment). IgG1 is most abundant in serum among the four IgG subclasses (IgG1, 2, 3 and 4) and binds to Fc receptors (FcγR ) on phagocytic cells with high affinity. Fc fragment is demonstrated to mediate phagocytosis, trigger inflammation, and target Ig to particular tissues. Protein G or Protein A on the surface of certain Staphylococcal and Streptococcal strains specifically binds with the Fc region of IgGs, and has numerous applications in biotechnology as a reagent for affinity purification. Recombinant IgG Fc Region is suggested to represent a potential anti-inflammatory drug for treatment of human autoimmune diseases.
Carosella ED. et al., 1988, Cell Immunol. 112: 262-70.
Eckle I. et al., 1990, Clin Exp Immunol. 81: 352-6.
Lorriaine C. et al., 1995, J Biol Chem. 270:8164-71.
Gomez-Guerrero C. et al., 2000, J Immunol. 164: 2092-101.
Sprague ER. et al., 2004, J Biol Chem. 14: 14184-93.