Mouse igg magnetic beads

Freshly lysed parasites were harvested, filter-purified and centrifugated at 1000 × g for 10 minutes. Parasites were resuspended in 1 ml of PBS with 1% formaldehyde and incubate at room temperature with gentle agitation for 10 minutes. Parasites were centrifugated and resuspended with 0.125 M glycine solution in PBS and incubated at room temperature for 5 minutes to stop the cross-linking reaction. Parasites were centrifugated and washed with PBS one time and then resuspended with IP lysis buffer (25 Mm Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40 and 5% glycerol) and incubated at room temperature for 1 hour with rocking. The lysate was then centrifugated at maximum speed (~ 20,000 xg) for 10 minutes. The supernatant was collected and precleaned by incubation with 25 ul of mouse IgG magnetic beads (Cell signaling) for 1 hour at room temperature. The unbound lysate was isolated from the IgG beads by using a magnetic bead rack and were incubated with 25 ul of anti-HA magnetic beads (Fisher Scientific) for 1 hour at room temperature. Both mouse IgG (used as a control) and anti-HA magnetic beads were washed with IP lysis buffer for three times and with PBS for another three times.
For mass spectrometry analysis, samples were submitted to the Indiana University School of Medicine Proteomics Core facility for protein identification by mass spectrometry as described before⁵³ .

As described previously (22 (link)), freshly lysed parasites were harvested and incubated for 10 min in PBS with 5 mM DSSO. Tris buffer was added to a final concentration of 20 mM to quench the reaction at room temperature for 5 min. After washes, the parasites were lysed with 1 mL RIPA lysis buffer containing a protease and phosphatase inhibitor cocktail at 4°C for 1 h. The lysate was then centrifuged, and the supernatant was incubated with 25 μL of mouse IgG magnetic beads (Cell Signaling) for 1 h at room temperature for precleaning. The unbound lysate was separated from the IgG beads and was incubated with 25 μL of anti-HA magnetic beads (Fisher Scientific) for another hour at room temperature. The anti-HA magnetic beads were washed with RIPA lysis buffer and PBS and then submitted to the Indiana University School of Medicine Proteomics Core facility for mass spectrometry.

Cells for co-immunoprecipitation assay were lysed in Pierce IP lysis buffer (Thermo Scientific, Cat No. 87787) containing a protease inhibitor cocktail. For immunoprecipitation of FLAG or MycTag, the whole cell lysates were incubated with anti-Flag magnetic beads (Bimake, Cat No. B26101), anti-Myc magnetic beads (Bimake, Cat No. B26301) or Mouse IgG Magnetic beads (Cell Signaling Technology, Cat No. #5873) respectively overnight at 4 °C with rotation. After washing three times with PBST buffer, the immune complexes were boiled in SDS sample loading buffer and subjected to western blot analysis.