This Mouse MD1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of MD1 protein (Cat: 50062-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain (Met 1-Ser 162) of mouse LY86 (NP_034875.1) precursor was expressed with a C-terminal polyhistidine tag.
The secreted recombinant mouse LY86 consists of 166 amino acids and has a calculated molecular mass of 18 kDa. As a result of glycosylation, the apparent molecualr mass of rmLY86 is approximately 24-30 kDa protein in SDS-PAGE under reducing conditions.
Mouse MD1 HEK293 Overexpression Lysate: 使用指南
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.
MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 15 (RP15) which is a molecule containing leucine-rich repeats and is expressed on B cells, dendritic cells, and macrophages, while MD-2 is associated with TLR4. MD-1 is required for efficient RP15 cell surface expression and function. It is indicated that the RP15/MD1 complex, in conjunction with TLR4, mediates the innate immune response to LPS in B cells, and also plays a role in protecting against apoptosis, B-cell proliferation, etc. Mouse MD-1 cDNA encodes a 162 amino acid precursor protein with a putative 19 aa signal peptide and two potential N-linked glycosylation sites. It shares 4% and 66% amino acid sequence identity with chicken and human MD-1 respectively. MD-1 is mainly expressed in the spleen, and also detectable in the liver, brain, thymus, and kidney.
lymphocyte antigen 86
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