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Figure 3 | Chemistry Central Journal

Figure 3

From: Development and immunochemical evaluation of a novel chicken IgY antibody specific for KLK6

Figure 3

Sensitivity and specificity of IgYs. Recombinant and endogenous KLK6 were detected by Western blotting using IgYs at 1:2,500. A, IgYs could detect as low as 1 ng (30 fmol) of rKLK6, while the widely used anti-KLK6 rabbit polyclonal [30] was limited to 10 ng (300 fmol). B, IgYs could recognize rKLK6 (100 ng) but lacked crossreactivity with its most homologous KLK13 (rKLK13, 1.5 μg) but also KLK5 (rKLK5, 1 μg) (Western blot, upper). Integrity of the loaded proteins was confirmed by Coomassie staining (SDS-PAGE gel, lower). C, IgYs lack crossreactivity with any of the secreted proteins as no band could be detected in MDA-MB-231 cells that do not produce KLK6 (lane 1, left). A single band of 37 kDa corresponding to endogenous KLK6 could be detected in MDA-MB-468 and MDA-MB-231 C5WT, a cDNA-transfected clone to stably express KLK6 [18] (lanes 2 and 3 respectively; left). Moreover, lack of crossreactivity of IgYs with other cellular proteins was confirmed by analysis of whole cell lysates (100 μg) isolated from MDA-MB-231 C5WT (lane 1, right) and MDA-MB-468 (lane 3, right). Additionally, IgYs could recognize a mutant form of KLK6 produced by MDA-MB-231 C7MS transfectants (lane 2, right) that lacks enzymatic activity [18]. D, Heat-induced denaturation of rKLK6 increases the sensitivity of IgYs. 5, 10 and 20 min refer to times of incubation at 95°C, while 5 and 10 sec denote the time of exposure; the two bands correspond to glycosylated (31 kDa) and non-glycosylated (25 kDa) rKLK6 produced in Pichia pastoris.

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