2 M��cm (Millipore). Reagents and solvents were of analytical purity ref 3 and used without any purification steps. Experiments were carried out at room temperature Inhibitors,Modulators,Libraries unless stated otherwise.2.2. Preparation of F(ab��) fragmentsAccording to the manufacturer��s instructions a portion of papain agarose from papaya latex (Sigma) was suspended in 400 ��L of H2O and incubated at 4 ��C for 2 hours, stirring every 15 minutes. Subsequently, the suspension was centrifuged for 5 min (20 ��C, 600 g). Then the papain agarose was rinsed twice in the activation buffer (50 mM Na2HPO4, 20 mM l-cysteine, 1 mM EDTA, pH 7) and activated in the same buffer at 37 ��C for 20 min with shaking. Further the papain agarose was washed three times with 3 �� digestion buffer (150 mM Na2HPO4, 3 mM EDTA, pH 7.

0), and suspended in 2 �� digesting buffer (100 mM Na2HPO4, 2 mM EDTA, pH 7.0) to the final concentration of 1 ��g/��L. This pre-activated papain agarose was used to prepare F(ab��) fragments from Anti-His(C-term) antibody (Invitrogen). IgG (6 ��g) was digested in 1x digestion buffer (50 mM Na2HPO4, 1 mM Inhibitors,Modulators,Libraries EDTA, pH 7.0; 20 ��L) at 37 ��C for 1 hour with constant shaking. The ratio of papain agarose to IgG was about 1:2 (w/w). The digestion was finished by centrifugation (5 minutes, 4 ��C, 1,000 g) and the supernatant was used for polyacrylamide gel analysis (Figure S1) and preparation of immunosensor.2.3. Preparation of antigens (rSPI2 proteins)SPI2 cDNA was cloned into the pPICZ��B plasmid carrying the sequences encoding of myc epitope and polyhistidine tag.

Transformation of Pichia pastoris cells (SMD 1168) by the pPICZ��B/SPI2 plasmid and the production of rSPI2-His6 protein was done as described previously [26, 27]. Four days after induction of rSPI2-His6 expression, the culture medium was collected by centrifugation. rSPI2-His6 was purified, from Inhibitors,Modulators,Libraries the culture medium, on Ni-NTA-agarose with >95% purity following the procedure described by K?udkiewicz et al. [27]. The culture medium without expressed protein (Pichia cells transformed with the pPICZ��B plasmid) was used as a control medium. The recombinant SPI2 protein without histidine-tag (rSPI2) was obtained according to the procedure described previously [26,27].2.4. Fabrication of gold nanaorods (GNR)Gold nanorods were fabricated according to a reported procedure [28]. For gold-seed production, 0.5 mL of 0.

01 M HAuCl4 trihydrate solution in water and 0.5 mL of 0.01 M sodium citrate solution in water were added to 18 mL of deionized water and stirred. Next, 0.5 mL of freshly prepared 0.1 M NaBH4 was added and the solution color changed from colorless to orange. Stirring was stopped and the solution was left undisturbed Inhibitors,Modulators,Libraries Drug_discovery for 2 hours. The resulting selleck kinase inhibitor spherical nanoparticles were used as gold seed. For gold nanorod growth from the seeds, three flasks were labeled A, B and C. Growth solutions A and B consisted of 9 mL of 0.1 M cetyltrimethylammonium bromide (CTAB) in water, 0.25 mL of 0.01 M HAuCl4 trihydrate in water, 50 ��L of 0.