Lymphocyte Protein Expression Analysis

Lymphocytes were isolated from the blood by the method describe previously by Dey et al. [19 (link)]. In brief, after sonication, peripheral blood lymphocytes (PBLs) isolated from case and control were used for immunoblotting. Protein content of the case and control samples was estimated by the method describe by Lowry [20 (link)]. The blood lymphocytes were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE; 3% stacking gel and 7.5% separating gel) and electroblotted on Immobilon-P membrane (Millipore, USA). The membranes were incubated with the primary antibodies raised against ASPN [21 (link)] and COMP [22 (link)] (1:1000 dilution) in 5 ml of phosphate-buffered saline containing 0.02% Tween-20 and 0.02% sodium azide, PBST for 3 h at room temperature. The membranes carrying lymphocytes were incubated with 1:2000 dilution of rabbit anti-rabbit IgG-horseradish peroxidase (secondary antibody). Following incubation, membranes were washed five times with PBST (5–10 min each) and then processed for detection with chemiluminescence substrate. The membranes were visualized on VERSA DOC Imaging System (Model 1000, Bio-Rad, USA) and quantitative analysis using Quantity One Quantitation software (Bio-Rad). Prior to studying protein expression of these genes, normalization of proteins in the lymphocytes was carried out using beta-actin antibody.