Single and double stranded calf thymus dna

In-vitro formation of a complex between PfCoSP, RNA/DNA and alpha/beta tubulin were tested using cellulose beads with immobilized single and double stranded calf thymus DNA (Sigma Aldrich, USA). 1 μg of PfCoSP was mixed with 500 μL cellulose matrix (1 mg/mL) and incubated at 4°C for 30 min. Bead pellet was extensively washed with 200 μL buffer followed by incubation with 5 μg recombinant purified alpha/beta tubulin. After washing, beads were boiled in 1X SDS-PAGE sample loading dye before loading on 12% SDS-PAGE. Immunoblotting was performed to probe for PfCoSP and alpha/beta tubulin using anti- PfCoSP and anti-alpha/beta tubulin antibodies respectively on the same blot. PfCoSP was incubated with cellulose beads with immobilized single and double stranded calf thymus DNA separately as positive controls. BSA was incubated with beads separately and incubated with alpha/beta tubulin as negative controls.

The nucleic acid binding property of protein was evaluated using cellulose beads with immobilized single and double stranded calf thymus DNA (Sigma Aldrich, USA). Here, 1 μg of recombinant PfCoSP was mixed with 500 μL cellulose matrix (1 mg/mL) and incubated at 4°C for 30 min. 200 μL buffer was used to wash bead pellet. Post washing, beads were boiled in 1X SDS-PAGE sample loading dye and loaded on 15% SDS-PAGE.
Cellulose beads with immobilized single and double stranded calf thymus DNA were also used to test whether LI71 can inhibit PfCoSP interaction with DNA/RNA. Here 500 μL cellulose matrix (1 mg/mL) was incubated with mixed stage parasite lysate treated with and without LI71 (1 μM) at 25°C and 37°C for 6 h. 200 μL buffer was used to wash bead pellet. Post washing, beads were boiled in 1X SDS-PAGE sample loading dye and loaded on 15% SDS-PAGE. Samples were subjected to western blotting and probed with anti-PfCoSP antibodies.