Monoclonal anti polyhistidine peroxidase conjugate

GST, GST-PfAKAL glutathione beads were incubated with (His)₆-Pf14-3-3I or (His)₆-PfPKA-R recombinant proteins. Also, GST and GST-PfPKA-R glutathione beads were incubated in (His)₆-Pf14-3-3I recombinant protein. Adenosine monophosphate (AMP) or cyclic AMP cross-linked agarose beads were incubated in recombinant (His)₆-PfAKAL protein solution for 2 h at 4 °C.
For all pull down experiments, beads were washed in RIPA buffer, and the protein complexes were analysed by SDS-PAGE and an anti-histidine western blot (Sigma Aldrich Monoclonal Anti-polyHistidine Peroxidase Conjugate) was performed to identify interactions.

Purified TgCBS and TgCBS R353* (100 μg) were cleaved with trypsin at 1:200 (w/w) ratio at 25 °C, in 20 mM sodium phosphate buffer pH 8.0 [39 (link)]. The proteolytic cleavage was stopped in 15 μL aliquots at time intervals of 0, 1, 5, 10, 20, 40, 60, 100, and 120 min by boiling the sample for 5 min. The aliquots were then subjected to SDS–PAGE and Western blot analysis by using the monoclonal anti-polyhistidine peroxidase conjugate (Sigma-Aldrich, Milan, Italy dilution 1:2000) against the N-terminal His-tag. The time-course of enzyme activity during trypsinolysis was determined using the CBL-LDH assay as described previously. To evaluate the sizes of the proteolytic fragment, 250 μg of TgCBS were incubated with trypsin for 120 min. The reaction was stopped by adding a two-fold weight excess of soybean trypsin inhibitor (Sigma) and the proteolysis product was loaded on a Superdex 200 10/300 GL column.