Human Tim4/TIMD4 HEK293 Overexpression Lysate

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Human Tim4/TIMD4 HEK293 Overexpression Lysate: Product Information

Product Description
This Human Tim4/TIMD4 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of Tim4/TIMD4 protein (Cat: 12161-H08H) from the overexpression lysate was verified.
Expression Host
HEK293 Cells
Species
Human
Sequence Information
A DNA sequence encoding the human TIMD4 (AAH08988.1) extracellular domain (Met 1-Leu 315) was expressed, fused with a polyhistidine tag at the C-terminus.
Molecule Mass
The recombinant human TIMD4 consists of 302 amino acids and has a predicted molecular mass of 32.7 kDa. In SDS-PAGE under reducing conditions, the apparent molecular mass of rhTIMD4 is approximately 60-65 kDa due to glycosylation.

Human Tim4/TIMD4 HEK293 Overexpression Lysate: Usage Guide

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.

Human Tim4/TIMD4 HEK293 Overexpression Lysate: Alternative Names

Human SMUCKLER Overexpression Lysate; Human TIM4 Overexpression Lysate

Tim4/TIMD4 Background Information

A type I transmembrane protein called TIM4 (T-cell immunoglobulin- and mucin-domain-containing molecule; also known as TIMD4), which belongs to the immunoglobulin superfamily and TIM family. TIM4 is involved in regulating T-cell proliferation and lymphotoxin signaling. It is a ligand for HAVCR1/TIMD1. Recent reports indicate that dendritic cell (DC)-derived T-cell immunoglobulin and mucin domain molecule (TIM)-4, which is expressed on dendritic cells and macrophages, plays an important role in the initiation of T(H)2 polarization. TIM4 bound apoptotic cells by recognizing phosphatidylserine via its immunoglobulin domain. The expression of TIM4 in fibroblasts enhanced their ability to engulf apoptotic cells. TIM4 is phosphatidylserine receptor for the engulfment of apoptotic cells, and may also be involved in intercellular signalling in which exosomes are involved. Modulation of TIM4 production in dendritic cells (DCs) represents a novel therapeutic approach for the treatment of peanut allergy. The interaction of TIM1/TIM4 played a critical role in sustaining the polarization status of Th2 cells in allergic rhinitis (AR) patients. Cross-linking FcgammaRI by antigen/IgG complexes increased the production of TIM4 by dendritic cells via upregulating tumor necrosis factor-alpha in DCs. Specific immunotherapy (SIT) suppresses the skewed Th2 responses via disrupting the interaction of TIM1/TIM4 in antigen-specific Th2 cells.
Full Name
T-cell immunoglobulin and mucin domain containing 4
References
  • Miyanishi M, et al. (2007) Identification of Tim4 as a phosphatidylserine receptor. Nature. 450(7168): 435-9.
  • Feng BS, et al. (2008) Disruption of T-cell immunoglobulin and mucin domain molecule (TIM)-1/TIM4 interaction as a therapeutic strategy in a dendritic cell-induced peanut allergy model. J Allergy Clin Immunol. 122(1): 55-61.
  • Cai PC, et al. (2009) Association of TIM4 promoter polymorphism -1419GA with childhood asthma in a Chinese Han population. Tissue Antigens. 74(1): 11-6.
  • Zhao CQ, et al. (2010) Specific immunotherapy suppresses Th2 responses via modulating TIM1/TIM4 interaction on dendritic cells. Allergy. 65(8): 986-95.
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