Western blot analysis of recombinant SMT3 (yeast SUMO) using SMT3-A1(Cys) as primary and anti-His-HRP as secondary


Overview
Abstract
This application describes the Western blot analysis of a recombinant SMT3 fusion protein spiked into an E.coli whole cell lysate in diminishing amounts using SMT3-A1(Cys) as primary and anti-His-HRP as secondary, and developing with TMB for membranes. The limit of detection of this colorimetric assay was between 3 and 6 ng.
Type
  • Western
Specimen
Target
Scores
Outcome
Successful
Signal
Acceptable
Cross Reactivity
Acceptable
Background
Acceptable
Dynamic Range
Acceptable
Results
Figure

Image 

Western blot of yeast SUMO with nanoCLAMP SMT3-A1

Legend 

Immunostaining of SMT3 fusion protein with SMT3-A1(Cys).  A recombinant SMT3 fusion protein (SMT3-NusA) was spiked into E.coli whole cell lysate, separated by SDS-PAGE, and transferred to PVDF (A) or stained with Coomassie (B). A: blot was probed with His-tagged SMT3-A1(Cys) at 0.25 ug/ml, followed by anti-His-HRP (1/4000), and detection with TMB for membranes (Thermo).