Immunoblot Validation of Ehrlichia Proteins

The expression of Ehrlichia proteins by IVTT was confirmed by dot immunoblot with horseradish peroxidase (HRP)-labeled mouse anti-His tag monoclonal antibody (1:500; GenScript) as described previously (Luo et al., 2020 (link)). The immunoreactivity of native and denatured proteins was also examined by dot immunoblot using IVTT-expressed proteins purified by MagneHis protein purification system (Promega, Madison, WI) according to the manufacturer. Immunoblots were probed with either HME or CME serum (1:200) and developed with TMB 1-component substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD).

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Luo T., Patel J.G., Zhang X, & McBride J.W. (2024). Antibody reactive immunomes of Ehrlichia chaffeensis and E. canis are diverse and defined by conformational antigenic determinants. Frontiers in Cellular and Infection Microbiology, 13, 1321291.

Publication 2023

horseradish peroxidase MagneHis protein purification system

Corresponding Organization : The University of Texas Medical Branch at Galveston

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Variable analysis

independent variables

Expression of Ehrlichia proteins by IVTT
Purification of IVTT-expressed proteins by MagneHis protein purification system

dependent variables

Immunoreactivity of native and denatured proteins examined by dot immunoblot

control variables

Horseradish peroxidase (HRP)-labeled mouse anti-His tag monoclonal antibody (1:500; GenScript) used for dot immunoblot
HME or CME serum (1:200) used for immunoblot probing
TMB 1-component substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD) used for immunoblot development

positive controls

Not explicitly mentioned

negative controls

Not explicitly mentioned

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