Team:HokkaidoU Japan/Protocols/Infection Assay

From 2011.igem.org

(Difference between revisions)
Sprkata (Talk | contribs)
(Created page with " {{Team:HokkaidoU_Japan/header}} =Preparation of T3SS ''E. coli''= =Infection assay using onion cells= =Infection assay using HeLa cells= =Detection of injected protein u...")
Newer edit →

Revision as of 02:01, 5 October 2011

Contents

Preparation of T3SS E. coli

Infection assay using onion cells

Infection assay using HeLa cells

Detection of injected protein using GSK tag

Protein extraction from infected HeLa cells

SDS-PAGE and Western Blot analysis

HeLa cell lysates were subjected to SDS-PAGE, and separated proteins were transferred to an Immobilon-P membranes (Millipore). The membranes were blocked with Blocking buffer (20 mM Tris, 150 mM NaCl, 0.05% Tween 20, 5% nonfat milk) for 1 h at room temperature. The blots were probed with Phospho-GSK-3β (Ser9) antibody (Cell Signaling Technology #9336) or GSK-3β antibody (Cell Signaling Technology #9315) diluted 1/1000 in Blocking buffer and incubated overnight at room temperature. Blots were washed three times with TTBS (20 mM Tris, 150 mM NaCl, 0.05% Tween 20) for 15 min each time. Secondary antibody (alkaline phosphatase-conjugated anti-rabbit immunoglobulin G) was diluted 1/1000 in Blocking buffer and incubated with the blots for 1.5 h at 37C. Blots were washed as described above and developed with BCIP/NBT.

Retrieved from "http://2011.igem.org/Team:HokkaidoU_Japan/Protocols/Infection_Assay"