Mouse TIM-1/KIM-1 HEK293 Overexpression Lysate

Product Description

This Mouse TIM-1/KIM-1 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of TIM-1/KIM-1 protein (Cat: 50321-M16H) from the overexpression lysate was verified.

Expression Host

HEK293 Cells

Species

Mouse

Sequence Information

A DNA sequence encoding the extracellular domain of mouse HAVCR1 isoform b (NP_001160104.1) (Tyr 22-Thr 212) was fused with a polyhistidine tag at the C-terminus and the Fc region of human IgG1 at the N-terminus.

Molecule Mass

The recombinant mouse HAVCR1/Fc is a disulfide-linked homodimer. The reduced monomer comprises 438 amino acids and predicts a molecular mass of 49 kDa. As a result of glycosylation, the apparent molecular mass of HAVCR1/Fc monomer is approximately 80-85 kDa in SDS-PAGE under reducing conditions.

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.