Human A33 HEK293 Overexpression Lysate: Product Information
This Human A33 overexpression lysate was created in HEK293 Cells and intented for use as a Western blot (WB) positive control. Purification of A33 protein (Cat: 11277-H08H) from the overexpression lysate was verified.
A DNA sequence encoding the extracellular domain of human GPA33 (Q99795-1) (Met 1-Val 235) was fused with a polyhistidine tag at the C-terminus.
The secreted recombinant human GPA33 consists of 225 amino acids and has a predicted molecular mass of 25 kDa. The apparent molecular mass of the rhGPA33 is approximately 35-40 kDa in SDS-PAGE under reducing conditions due to glycosylation.
Human A33 HEK293 Overexpression Lysate: Usage Guide
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.
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.
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.
Western Blot (WB) Optimal dilutions/concentrations should be determined by the end user.
Human A33 HEK293 Overexpression Lysate: Alternative Names
Human A33 Overexpression Lysate
A33 Background Information
Cell surface A33 antigen, also known as glycoprotein A33, is a single-pass type I membrane protein which is expressed in normal gastrointestinal epithelium and in 95% of colon cancers. GPA33 contains oneIg-like C2-type (immunoglobulin-like) domain and oneIg-like V-type (immunoglobulin-like) domain. The open reading frame encodes a 319-amino acid polypeptide having a putative secretory signal sequence and 3 potential glycosylation sites. The predicted mature protein has a 213-amino acid extracellular region, a single transmembrane domain, and a 62-amino acid intracellular tail. Intracellular traffic and recycling to the cell surface appear to play a major role in GPA33 function and to have an influence on its surface density superseding translational regulation. GPA33 has become a promising target of immunologic therapy strategies, but its biologic function and potential role in tumorigenesis are unknown. EpCAM protein and GPA33 mRNA expressions are specific and sensitive markers of Barrett's metaplasia (BM). GPA33 may also play a role in cell-cell recognition and signaling.
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