Fig. 2

Autoantibody response analysis in GBC using 1-D and 2-D immunoblotting. Pooled immunodepleted GBC tissue lysate was resolved by SDS–PAGE and electro-transferred to PVDF membrane followed by blocking and incubation with pooled plasma from healthy individuals (n = 11), gallstone cases (n = 11), GBC Stage I and II (n = 5), and GBC Stage IIIA (n = 9) (dilution 1:1000). The blots were then incubated with secondary antibody (anti-human IgG HRP conjugated, dilution 1:40,000) followed by development using ECL reagent. All images were acquired using Chemidoc MP (Bio-Rad). (a) Image analysis of 1-D immunoblots showed 03 protein bands (37 kDa, 30 kDa and 28 kDa) with high or specific immunoreactivity in GBC Stage I and II, and 02 protein bands (54 kDa and 39 kDa) with high or specific immunoreactivity in GBC Stage IIIA (encircled). (b) Coomassie-stained 2-D gel images and (c) 2-D immunoblot images were analyzed by PDQuest software (Bio-Rad) which showed 25 protein spots (marked with an arrow) with specific reactivity in GBC cases. The proteins showing non-specific reactivity both in cases and controls are marked by circle. The full-length blot images are presented in Supplementary Figure S3