DETECTION AND IDENTIFICATION OF D6 AND DUFFY ANTIGEN/RECEPTOR FOR CHEMOKINES (DARC) BY ANTIBODY BASED ASSAYS

Abstract

D6 and DARC are decoy chemokine binding receptors. They had been studied extensively in recent years for their role in breast cancer studies. The purpose of this study was to improve some critical steps in Western Blot in order to detect and identify recombinant D6 and DARC which has been expressed intracellularly by Pichia-GS115. Total proteins of Pichia-GS115 were obtained by breaking the yeast cells using acid-washed glass beads. Then, the lysates were treated with treatment buffer before electrophoresed on SDS-polyacrylamide gel. Western Blot was carried out after SDS PAGE to detect and identify the presence of recombinant D6 and DARC. Several parameters of Western Blot were studied and improved to enhance the specificity of results obtained. It was found that blocking of nitrocellulose membrane with 3% (w/v) skimmed milk at room temperature for 1 hour, incubation of primary antibody at the dilution of 1: 300 at room temperature for 16 hours and incubation of secondary antibody at the dilution of 1: 1000 at room temperature for 1 hour were able to enhance the sensitivity and specificity of Western Blot results. With the aid of prestained protein ladder as a marker, recombinant D6 and also DARC were shown as clear and precise bands at the range of molecular weight of 50-60 kDa on developed films using ECL substrate. The film exposure time was found to be 5 minutes for D6 and 10 minutes for DARC. As a result, the studies showed that improved Western Blot condition detected and identified the presence of recombinant D6 and DARC in the cell pellets of Pichia-GS115