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|Human Cell lysate that Mouse LY86 / MD1 transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|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.|
|Cell lysate was prepared by homogenization 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.|
|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.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|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. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
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 105 (RP105) which is a molecule containing leucine-rich repeats and is expressed on B cells, dentritic cells and macrophages, while MD-2 is associated with TLR4. MD-1 is required for efficient RP105 cell surface expression and function. It is indicated that the RP105/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 40% and 66% amino acid sequence identity with chicken and human MD-1 respectively. MD-1 is mainly expressed in spleen, and also detectable in liver, brain, thymus, and kidney.