This Mouse CLEC14A overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CLEC14A protein (Cat: 50304-M08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of mouse CLEC14A (NP_080085.3) (Met 1-Thr 386) was expressed, fused with a polyhistidine tag at the C-terminus.
The recombinant mouse CLEC14A consists of 376 amino acids and has a predicted molecular mass of 40.6 kDa. Due to glycosylation, rm CLEC14A migrates as an approximately 75 kDa band in SDS-PAGE under reducing conditions.
Mouse CLEC14A 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.
C-type lectin domain family 14 member A, also known as Epidermal growth factor receptor 5 and CLEC14A, is a member of the C-type lectin domain (CTLD) family that contains one c-type lectin domain and one EGF-like domain. Mouse CLEC14A is a 459 amino acid single-pass type I membrane protein. The superfamily of proteins containing C-type lectin-like domains (CTLDs) is a large group of extracellular Metazoan proteins with diverse functions. The CTLD structure has a characteristic double-loop ('loop-in-a-loop') stabilized by two highly conserved disulfide bridges located at the bases of the loops, as well as a set of conserved hydrophobic and polar interactions. Members of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily share a common fold and are involved in a variety of functions, such as generalized defense mechanisms against foreign agents, discrimination between healthy and pathogen-infected cells, and endocytosis and blood coagulation. Genome-level studies on human, elegans and melanogaster demonstrated almost complete divergence among invertebrate and mammalian families of CTLD-containing proteins (CTLDcps). The vertebrate CTLDcp families were essentially formed early in vertebrate evolution and are completely different from the invertebrate families. The composition of the CTLDcp superfamily in fish and mammals suggests that large scale duplication events played an important role in the evolution of vertebrates.
C-type lectin domain family 14, member A
Ebner S, et al. (2003) Evolutionary analysis reveals collective properties and specificity in the C-type lectin and lectin-like domain superfamily. Proteins. 53(1): 44-55.
Zelensky AN, et al. (2005) The C-type lectin-like domain superfamily. Gready JE. FEBS J. 272(24): 6179-217.