Rabbit anti protein a antibody

Immunoblotting and immunofluorescence were performed as previously described [51 (link)]. A rabbit anti-protein A antibody (Sigma-Aldrich) was used to detect TAP-tagging (1:40,000 dilution). TcDHH1 (47 kDa) was used as a control of the metacyclogenesis western blotting assay. Anti-TcDHH1 (1:100 dilution) was kindly provided by Jimena Ferreira da Costa. Bound antibodies were detected by the alkaline phosphatase method or by the Odyssey® imager (LI-COR) using the secondary antibodies IRDye 800CW or IRDye 680RD (1:15,000 dilution) [52 (link)]. Immunofluorescence slides were analyzed by inverted microscopy (Leica DMI6000B) associated with deconvolution software (Leica AF6000, microscope facility RPT07C PDTIS/ Carlos Chagas Institute - Fiocruz Paraná).

Protein lysates for Western blot were prepared by the TCA method. From overnight grown cultures, 3OD₆₀₀ equivalent cells were harvested, washed, and resuspended in 400 μl of 12.5% ice-cold TCA solution. The suspension was vortexed briefly and stored at −20°C for 12 h. The suspension was thawed on ice, pelleted at 14,000 rpm for 10 min, and washed twice with 350 μl of 80% acetone (ice cold). The washed pellets were air dried completely and resuspended in desired volume of lysis buffer (0.1N NaOH+1% SDS). Rabbit anti-Protein A antibody (P3775; Sigma-Aldrich) and the HRP-conjugated goat anti-rabbit secondary antibody, both were used at 1:5,000 dilution in 2.5% skim milk powder in 1XPBS. The blots were developed using chemiluminescent substrate (Bio-Rad) and imaged using Chemidoc system (Bio-Rad).

Western blotting was performed as previously described [21] (link). Briefly, 2×10⁶ procyclic form T. brucei clones at log phase of growth were harvested, resuspended in Laemli buffer, subsequently separated on a denaturing 11% SDS-PAGE gel. Proteins were transferred to a nylon Hybond P membrane (Amersham Pharmacia), incubated with a 1∶50000 dilution of rabbit anti-Protein A antibody in transfer buffer and probed for expression of TbSF3a60-TAP using the peroxidase-anti-peroxidase complex (1∶50,000, Polysciences, Inc. - www.polysciences.com). Subsequently, clones expressing C-terminally TAP-tagged SF3a60 were harvested and the subcellular localization of the tagged SF3a60 determined by indirect immunofluorescence [22] (link). To detect the over-expressed SF3a60-TAP, 10⁶ trypanosomes were sedimented, re-suspended, then fixed in 4% paraformaldehyde (w/v) in 1x PBS for 18 min, sedimented again for 2 min, re-suspended in phosphate buffered saline (PBS), and allowed to settle down on a poly-lysine-coated glass surface for 25 min. Cells were blocked with 0.5% gelatin/PBS and thereafter incubated with a 1∶50000 dilution of rabbit anti-Protein A antibody (Sigma) for 1 h and with a 1∶500 dilution of the second antibody Alexa Fluor 594 goat a- rabbit IgG (Molecular 25 probes) for 40 min. The kinetoplast and the nuclear DNA were then stained with 100 ng/ml DAPI/1x PBS for 10 min.