Strep tactin conjugated to horseradish peroxidase

Mycoplasma cell lysates were quantified using the Pierce ™ BCA Protein Assay Kit. Cell extracts were separated by electrophoresis using NuPage™ 4-12% Bis-Tris polyacrylamide gels (Invitrogen) and proteins transferred onto nitrocellulose membranes using an iBlot™ dry system (Invitrogen). Membranes were blocked in a PBS buffer containing 0.1% Tween 20 supplemented with 5% skim milk (Sigma) or 3% BSA in the case of Strep-tag detection. For immunodetection, membranes were probed with polyclonal anti-CAT (abcam, 1:2,000), monoclonal anti-FLAG M2 (Sigma, 1:5,000), Strep-Tactin conjugated to horseradish peroxidase (IBA lifesciences, 1:10,000), DsRed polyclonal antibody (Takara, 1:2,000), anti-polyclonal anti-ssrAmk tag (1:2,000) or polyclonal antibodies specific to mycoplasma proteins (kind gift of Dr. Herrmann, Heidelberg University). These include anti-Lon (1:3,000) and anti-FtsH (1:3,000), which were also used as loading controls together with anti-CAT (abcam, 1:2,000) antibody. Anti-mouse IgG (1:10,000) or anti-rabbit IgG (1:5000) conjugated to horseradish peroxidase (Sigma) were used as a secondary antibody. Blots were developed with the Supersignal™ West Femto or West Pico Chemiluminescent Substrate detection Kit (Thermo Scientific) and signals detected in a LAS-3000 Imaging System (Fujifilm).