Human CD32A HEK293 Overexpression Lysate

Product Description

This Human CD32A overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of CD32A protein (Cat: 10374-H27H1) from the overexpression lysate was verified.

Expression Host

HEK293 Cells

Species

Human

Sequence Information

A DNA sequence encoding the extracellular domain (Met 1-Met 210) of human CD32a (P12318-1) was fused with a c-terminal polyhistidine tagged AVI tag at the C-terminus. It is identical to FCGR2A131H/R in the reference.

Molecule Mass

The recombinant human CD32a consists of 213 amino acids after removal of the signal peptide and predicts a molecular mass of 23.6 KDa. It migrates as an approximately 32 KDa band due to glycosylation by 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.