Serum Biomarker Chip Datasheet - Whatman

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Two-Color Labeling & Detection Kit The Two-Color Labeling and Fluorescent Detection Kit is designed to label two protein samples. The labeled proteins are pooled and probed against arrayed antibodies in a competitive binding assay, and detected using indirect fluorescence. • Highly efficient and uniform labeling of complex serum samples • Reproducible labeling and signal detection • Stable, robust and fast non-enzymatic procedure • Reduces pH dependency of labeling efficiency • System solution includes labeling reagents, fluorescent conjugate and bench-friendly protocol The kit is intended for use with two-pad FAST Slides, including the Serum Biomarker Chip. The kit contains the Universal Linkage System (ULS) Labeling and Detection Procedure Overview chemistry to label samples containing approximately 250 µg of protein in serum, plasma, or a whole cell lysate. The kit is designed to label two different protein samples, each with a different hapten. Sufficient labeling reagent is provided to perform a hapten swapping experiment. Benefits of the two-color dye-swap assay • Accounts for hapten-specific differences in either Biotin-ULS or Fluorescein- ULS labeling efficiencies • Averages differences in antibodyantigen binding interactions caused by steric hindrance • Minimizes chip-to-chip variability – includes an internal control within the assay The first pad on the slide is probed with a mixture of two different protein samples, each labeled with a different hapten; the second pad is probed with the same two protein samples but with the haptens reversed. The normalized intensity for each Sample A Sample B Sample A Sample B Block slide with S&S Blocking Buffer for 30 min at room temperature Mix 1 Mix 2 element of each pad is calculated as the average of the biotin- and fluoresceinlabeled derived intensities from a two-pad experiment. The ratio between the signal intensity at each spot corresponds to the concentration ratio of the proteins found in the two samples. This method is attractive for antibody chips as it takes into account any hapten-specific differences in antigenantibody interactions. The use of the ULS labeling system minimizes background by using indirect fluorescence detection, labels multiple amino acids, and requires no additional materials or reagents. Label serum proteins with Biotin-ULS ( ) and Fluorescein-ULS ( ) for 2-6 hours at 42 °C Terminate reaction and remove unbound ULS reagent with spin columns Pool Samples Incubate labeled proteins with Serum Biomarker Chip for 4 hours to overnight at room temperature W H A T M A N S E R U M B I O M A R K E R C H I P D A T A S H E E T Wash 6X to remove unbound proteins
APPLICATION DATA Serum protein profiles of cancer patients. Serum proteins from cancer patients and corresponding age- and gender-matched controls were labeled with Biotin-ULS and Fluorescein- ULS, respectively, then pooled and probed against the Serum Biomarker Chip. The competitive binding was detected using both streptavidin-Dy647 and anti-fluorescein antibody-Dy547 conjugates. Spot-finding, background corrections, and two-channel data sampling was done using ArrayVision FAST ® software. The SBC Analysis Workbook is a Microsoft ® Excel file that converts fluorescent data into numerical values that represent the abundance of antigen in Sample A relative to Sample B. The average spot intensities for each analyte were plotted to reveal the over- and under-abundant serum biomarkers in the cancer patient (y-axis) compared to serum of a healthy individual (x-axis) for bladder, breast, colon, and prostate cancer. Upper and lower purple control lines represent 1.25 standard deviations above and below a ratio of 1 (no change in protein expression). Based on previous experimental results with the same serum labeled with both haptens and competed against itself, we recommend that only ratios greater than 1.25 std devs on either side of ratio=1 be considered significant expression level differences (these values lie outside the normal noise level of the assay). Ratios are plotted on a log 2 scale so that a value of 0 represents no change in expression between the 2 samples, with ratios representing up and down regulation being distributed equally around 0. Incubate Serum Biomarker Chip with two-color fluorescent detection solution for 1 hour at room temperature Log2 Ratio Log2 Ratio Log2 Ratio Log2 Ratio 0.40 0.30 0.20 0.10 0.00 -0.10 -0.20 -0.30 -0.40 -0.50 2.00 1.50 1.00 0.50 0.00 -0.50 -1.00 -1.50 1.50 1.00 0.50 0.00 -0.50 -1.00 -1.50 -2.00 0.50 0.40 0.30 0.20 0.10 0.00 -0.10 -0.20 Wash 6X to remove unbound proteins Excite 547 nm Bladder Cancer Breast Cancer Colon Cancer Prostate Cancer Scan at two wavelengths, spot-find, and sample data Excite 647 nm W H A T M A N S E R U M B I O M A R K E R C H I P D A T A S H E E T Log2 Ratio 0.50 0.40 0.30 0.20 0.10 0.00 -0.10 -0.20 Prostate Cancer Bioinformatic analysis
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Serum Biomarker Chip Datasheet - Whatman

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