Both IFA and WB analyses were performed as described previously (Durante et al. 2015 (link)) and the tagged proteins were visualized using primary monoclonal α-V5 (Thermo Fischer Scientific; 1:500 for IFA and 1:2,000 for WB), α-cMyc, and α-HA (both from Sigma-Aldrich; 1:2,000 for WB) antibodies, followed by secondary donkey α-mouse Alexa 488 antibody (Molecular Probes) for IFA and peroxidase-coupled rabbit α-mouse antibody (Sigma-Aldrich) for WB. Micrographs were taken with a Zeiss Axioplan-2 fluorescence microscope (Zeiss) and the acquired images were processed with ImageJ software (Schindelin et al. 2012 (link)).
Protocol detail
Visualizing Tagged Proteins by IFA and WB
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Antibodies
Antibody
Donkey
Fluorescence microscope
Molecular probes
Mouse
Peroxidase
Proteins
Rabbit
Corresponding Organization :
Other organizations : Czech Academy of Sciences, Biology Centre, Institute of Parasitology, University of Ostrava, University of South Bohemia in České Budějovice, Sechenov University
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Variable analysis
independent variables
- None explicitly mentioned
- Visualization of tagged proteins using primary monoclonal α-V5, α-cMyc, and α-HA antibodies
- None explicitly mentioned
- Positive control: Not specified
- Negative control: Not specified
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