Western blot analysis of recombinant NusA using nusA-A1(Cys) as primary and anti-His-HRP as secondary


Overview
Abstract
This application describes the Western blot analysis of a recombinant NusA fusion protein spiked into an E.coli whole cell lysate in diminishing amounts using nusA-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 12 and 6 ng.
Type
  • Western
Specimen
E. coli BL21(DE3)
Target
NusA
Scores
Outcome
Successful
Signal
Acceptable
Cross Reactivity
Acceptable
Background
Acceptable
Dynamic Range
Acceptable
Results
Figure

Image 

Western blot of NusA with nanoCLAMP nusA-A1

Legend 

Immunostaining of NusA fusion protein with nusA-A1(Cys).  A recombinant NusA 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 nusA-A1(Cys) at 0.25 ug/ml, followed by anti-His-HRP (1/4000), and detection with TMB for membranes (Sigma).
Methods
Protocol
Western blot using nanoCLAMP and anti-His-HRP
Product Lot(s) Used
Lot Function Academic Price Commercial Price Order Equivalent Product
nC-nusA-A1(Cys)10nmol-23 Detection $275 $525
Variant(s) Used
VariantFunction
nanoCLAMP nusA-A1(Cys)
  • Detection