Product Description
This Human LAG3 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of LAG3 protein (Cat: 16498-H05H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Sequence Information
A DNA sequence encoding the human LAG3 (NP_002277.4) (Met1-Leu450) was expressed with the Fc region of mouse IgG1 at the C-terminus.
Molecule Mass
The recombinant human LAG3 consists of 656 amino acids and predicts a molecular mass of 72 kDa.
Preparation Method
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.
Lysis Buffer
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.
Recommend Usage
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.
Sample Buffer
1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).
Stability & Storage
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.
Application
Western Blot (WB)
Optimal dilutions/concentrations should be determined by the end user.
LAG3, also known as CD223 and Lymphocyte activation gene 3, belongs to immunoglobulin (Ig) superfamily. LAG3 contains 4 extracellular Ig-like domains. The LAG3 gene contains 8 exons. It is selectively expressed in activated T and NK cells. LAG3 has a negative regulatory function in T cells. It also acts as as a new marker of T cell induced B cell activation. As a soluble molecule, LAG3 activates antigen-presenting cells through MHC class II signalling, leading to increased antigen-specific T-cell responses in vivo.
Immune Checkpoint Immune Checkpoint Targets Co-inhibitory Immune Checkpoint Targets Immunotherapy Cancer Immunotherapy Targeted Therapy References
Sigrid Hannier. et al., 1998, The Journal of Immunology. 161(8): 4058-65. Triebel F. et al., 1990, J Exp Med. 171 (5): 1393-405. Baixeras E. et al., 1992, J Exp Med. 176 (2): 327-37. Huard B. et al., 1997, Proc Natl Acad Sci. 94 (11): 5744-9. Kisielow M. et al., 2005, Eur J Immunol. 35 (7): 2081-8.