PS and MS thank DST for INSPIRE-Faculty award and FAST TRACK fellowship respectively. presence of liquid nitrogen in a ventilated hood. The pulverized samples were mixed in extraction answer made up of 0.2 M sodium chloride and 50 mM sodium phosphate buffer pH 7.2 and stirred for 24 h at 277 K. 2.5 g polyvinylpyrolidine (PVP) was mixed with 100 ml of the above solution and was further homogenized. The homogenate obtained was centrifuged at 277 K at 5000 g for 30 min. The protein was precipitated with 80% saturated ammonium sulfate. The precipitated protein was mixed in 50 mM sodium phosphate buffer pH 7.2. The proteins Tebuconazole present in the solution were examined in a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Physique 1). The protein solution was loaded onto a DEAE-Sephadex A-50 column (50 2 cm) equilibrated with 50 mM sodium phosphate buffer pH 7.2. The proteins were eluted using a continuous gradient of 0.0-0.5 M NaCl in the same Tebuconazole buffer in which two peaks were collected and pooled separately. The second peak containing the low molecular weight protein was loaded onto a Sephadex G-50 column (150 1 cm) equilibrated with 25 mM Tris-HCl, pH 8.0 and proteins were eluted using the same buffer. The fractions corresponding to a molecular mass of 13 kDa were pooled, collected, dialyzed and lyophilized. The purity of the sample was checked by SDS-PAGE. Open in a separate window Physique 1 SDS-PAGE showing proteins present in extracts of after ammonium sulphate precipitation. Lane A is usually protein molecular excess weight marker and Lane B, C, and D are showing protein bands in extract from after gel filtration chromatography. Allergenicity test To obtain the best conditions for activation in freshly isolated PBMCs derived from peripheral Tebuconazole bloods of healthy donor in our study, we performed a dose and kinetic response Tebuconazole of our novel protein for cytokines (IFN-) secretion by circulation cytometry. We found 24 hour of culture and 10 g/ml dose of Narcin were optimal for the cytokine response. PBMCs (0.5 106 /ml) were cultured for 24 hours with Golgi bodies transfer blocker in presence Narcin (10 g/ml) and with media alone (total RPMI and Golgi bodies transfer blocker (10 g/ml)). Isotype controls have been used to confirm the specificity of main antibody binding and to avoid any nonspecific antibody binding. Cultured cells were washed and surface stained with anti-CD4 and followed by IFN-, IL-10, IL-4 and IL-13 intracellular staining. We first examined frequency of IFN- and IL-10 production by CD4+ T cells in response to activation by Narcin (Physique 3A). Upon Narcin activation, the frequency of IFN- was 3.4 1.9 (p = 0.043) compared to un-stimulated cells producing IFN- at a frequency of 1 1.1 0.43 (Determine 3B). The percentage of IL-10 positive cells was also found to be 1.8 0.21 (p = 0.0001) when stimulated by the protein in contrast to 0.31 0.08 in un-induced cells (Determine 3B). We also observed a profound increase in the frequency of dual cytokines (DP = Rabbit polyclonal to ALKBH1 both IFN- and IL-10) generating CD4+ T cells in response to our novel allergen. Open in a separate window Physique 3 Effect of Narcin on cytokine production. A: Representative FACS plot showing percentage production of IFN-, IL-10 (upper panel) and IL-4 and IL-13 (lower panel) on gated CD4+ T cells. B: Data presents the mean S.D. for five individual experiments (n = 5). *Represents.