Horseradish peroxidase labeled antirabbit antibody

The cells were washed 2 times in cold PBS and then centrifuged for 10 minutes. Cells pellet were suspended in 10 µL per 2 × 10 6 cell/mL pro prep protein extraction buffer. Incubated on ice for 10 minutes, and then centrifuged at 3000 g for 15 minutes at 4 ℃. The supernatant was then transferred to a new tube and used for assay. The total protein concentration was determined by the Bradford method using a Bradford solution (Sigma Co.). The prepared protein were used for western blot analysis. Expression of MIF, IL-2 and IL-8 protein was quantified by western blot analysis. Proteins (20 µg/sample) were fractionated on a 15% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories Inc.) and transferred onto a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% skim milk (Bio-rad Co.), and then incubated with a primary antibody, anti-human IL-8, MIF, IL-2 (1 : 500; R&D systems). After washing, membranes were incubated with 1 : 1000 horseradish peroxidase-labeled antirabbit antibody (R&D systems) as the secondary antibody. The proteins were detected using ECL (Cyanagen) chemiluminescence kit.

The cells were washed 2 times in cold PBS and then centrifuged for 10 minutes. Cell pellets were suspended in 10 μL per 2 × 10 6 cell/mL pro-prep TM protein extraction buffer. Incubated on ice for 10 minutes, and then centrifuged at 3,000 × g for 15 minutes at 4℃. The supernatant was then transferred to a new tube and used for the assay. The total protein concentration was determined by the Bradford method using a Bradford solution (Sigma Co.). The prepared protein was used for western blot analysis. The expression of IL-2 protein was quanti ed by western blot analysis. Proteins (20μg/sample) were fractionated on a 15% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories Inc.) and transferred onto a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% skim milk (Bio-rad Co.) and then incubated with a primary antibody, anti-human IL-2 (1:500; R&D systems). After washing, membranes were incubated with 1:1000 horseradish peroxidase-labeled anti-rabbit antibody (R&D systems) as the secondary antibody. The proteins were detected using ECL (Cyanagen) chemiluminescence kit.

The cells were washed 2 times in cold PBS and then centrifuged for 10 minutes. Cells pellet were suspended in 10 μL per 2 × 10 6 cell/mL pro prep protein extraction buffer. Incubated on ice for 10 minutes, and then centrifuged at 3,000 × g for 15 minutes at 4℃. The supernatant was then transferred to a new tube and used for assay. The total protein concentration was determined by the Bradford method using a Bradford solution (Sigma Co.). The prepared protein were used for western blot analysis. Expression of iNOs, MIF, IL-2 and IL-8 protein was quanti ed by western blot analysis. Proteins (20μg/sample) were fractionated on a 15% sodium dodecyl sulfate-polyacrylamide gel (Bio-Rad Laboratories Inc.) and transferred onto a nitrocellulose membrane. Membranes were blocked for 1 hour in 5% skim milk (Bio-rad Co.), and then incubated with a primary antibody, anti-human IL-8, iNOS, MIF, IL-2 (1:500; R&D systems). After washing, membranes were incubated with 1:1000 horseradish peroxidaselabeled anti-rabbit antibody (R&D systems) as the secondary antibody. The proteins were detected using ECL (Cyanagen) chemiluminescence kit.