Figure 4.

(A) SDS-PAGE analysis of purified recombinant PgHsp70 protein. PgHsp70 carrying a N-terminal hexahistidine tag was overproduced in pET-28a(+) and purified by Ni²⁺ NTA affinity chromatography. Lane 1 is molecular mass markers; the sizes (in kDa) are indicated adjacent (left side) to the gel. Lane 2 IPTG-induced supernatant fraction containing enriched PgHsp70 protein (70 kDa). Lane 3 is un-induced protein PgHsp70, Lane 4 is Ni₂₊ NTA purified PgHsp70. The gel containing lanes 1–4 is stained with Coomassie Brilliant Blue. Lane 5: the purified PgHsp70 protein was run on another gel and silver stained. (B) Reconfirmation of PgHsp70 purity by the reverse-phase-HPLC analysis (detector: 280 nm) on C18 (Phenomenex, C18, 5 μM 1.D. 250 × 4.6 nm) using aceto-nitrile (0.1% TFA)/water (0.1% TFA) gradient 0−15 min at the rate of 2% min⁻¹ solvent; 15 to 45 min 0.5% min⁻¹ solvent at a flow rate of 1.0 ml min⁻¹.