2.5 ǀ Expression of cpOAS1 protein using western blotting
Caprine OAS1 proteins expressed in goat endometrial cells were separated
on a 12% SDS-PAGE gel and transferred electrophoretically to a PVDF
membrane (BiotraceTM PVDF, PALL Corporation, India) at
100 V for 2 h uteri as described earlier58. After
transfer, the membrane was incubated in blocking buffer [5% (w/v)
skim milk powder in PBS, pH 7.4] at room temperature for 1h with
gentle agitation. To detect the cpOAS1 isoform proteins expressed in the
goat endometrium, the membranes were incubated with rabbit OAS1
polyclonal antibody (Invitrogen, Catalog # PA5-41755) in 1:800
dilutions in blocking buffer at 4˚C overnight. To detect overexpressed
cpOAS1 isoforms, the membranes were incubated at room temperature for
1.5 h with HRP conjugated mouse anti-rabbit secondary antibodies (Santa
Cruz, CA, USA) to a 1:2000 dilution of in PBS. DAB substrate buffer
system (GeNei, India) was used to detect the presence of secondary
antibody. The densitometric analysis of protein was performed by NIH
ImageJ 1.44p software (National Institutes of Health, Bethesda,
Maryland, USA). The relative intensity of the bands was quantified and
normalized with β-actin (Santa Cruz, CA, USA).