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1. Il Introduction Cytokines play an important role in innate immunity apoptosis angiogenesis cell growth and differentiation They are involved in interactions between different cell types cellular responses to environmental conditions and maintenance of homeostasis In addition cytokines are also involved in most disease processes including cancer and cardiac diseases RayBio G Series Arrays are glass slide based antibody arrays which allow researchers to conduct rapid accurate expression profiling of hundreds of cytokines chemokines growth factors proteases soluble receptors and other proteins from any biological fluid Like a traditional sandwich based ELISA this array uses a matched pair of cytokine specific antibodies for detection After incubation with the sample the target cytokines are captured by the antibodies printed on the solid surface A second biotin labeled detection antibody is then added which recognizes a different epitope of the target cytokine The cytokine antibody biotin complex can then be visualized through the addition of the streptavidin conjugated Cy3 equivalent dye Like the Quantibody arrays G Series utilizes a highly sensitive and stable fluorescent readout which can be detected by most laser fluorescent scanner systems After capturing the spot densities with a laser scanner normalization of the raw data can be easily calculated by the researcher or by a quick copy paste into our excel b2. about 30 ml to cover the whole slide and then gently shake at room temperature for 15 minutes Decant Wash Buffer I Wash with 1x Wash Buffer II about 30 ml and gently shake at room temperature for 5 minutes Remove water droplets completely by gently applying suction with a pipette to remove water droplets Do not touch the array only the sides You may also dry the glass slide by a compressed N2 stream Imaging The signals can be visualized through use of a laser scanner equipped with a Cy3 wavelength green channel such as Axon GenePix In case the signal intensity for different cytokine varies greatly in the same array we recommend using multiple scans with a higher PMT for low signal cytokines and a low PMT for high signal cytokines F Data Analysis 15 Data extraction can be done using the GAL file that is specific for this array along with the microarray analysis software GenePix ScanArray Express ArrayVision MicroVigene etc GAL files can be found here www RayBiotech com Gal Files html Need help analyzing all that data Copy and paste your data into the Q Analyzer Tool specific for this array catalog number GSH ANG 2000 SW More information can be found in Section X Experiment 5 Image Scan A list of compatible laser scanners can be found here RayBiotech com Scanners 5 Data Extraction GenePix etc Analyze Results Analysis Tool sold separately see Section X IX Array
3. G Series Human Angiogenesis Array 2000 A combination of 2 non overlapping arrays to measure the relative expression levels of 60 human angiogenic proteins Catalog GSH ANG 2000 User Manual Last revised May 2015 Caution Extraordinarily useful information enclosed Ke RayBiotech The protein array pioneer ISO 13485 Certified 3607 Parkway Lane Suite 100 Norcross GA 30092 Tel 1 888 494 8555 Toll Free or 770 729 2992 Fax 770 206 2393 Web www RayBiotech com Email info raybiotech com 1 mm D O O O D mia Page 2 Additional Materials Required TETT VI Vil General Considerations A Sample Preparation B Handling Glass Slides C Incubation Protocol A Completely Air Dry The Glass Slide B Blocking amp Incubation C Incubation with Biotinylated Antibody Cocktail amp Wash D Incubation with Cy3 Equivalent Dye Streptavidin amp Wash E Fluorescence Detection F Data Analysis CO E ll Select Publications Ni E X X N NN q q qq gt Q Q O gt Q N oO Please read the entire manual carefully before starting your experiment l Overview Cytokines Detected 60 Arrays Included GSH ANG 2 30 GSH ANG 3 30 See Section IX for Array Map One standard glass slide is spotted with 16 wells of identical cytokine antibody arrays Each antibody is arrayed in quadruplicate Method list of compatible laser scanners
4. Map 11 QAH ANG 2 QAH ANG 3 Each antibody is printed in quadruplicate horizontally Each antibody is printed in quadruplicate horizontally 1 2 3 4 1 2 3 4 1 2 3 4 2 3 4 1 2 3 4 1 2 3 4 A POS1 POS2 NEG A POS1 POS2 NEG B Activin A AgRP Angiogenin B Angiogenin Angiostatin CXCL16 Cc Angiopoietin 2 ANGPTL4 bFGF Cc EGF FGF 4 Follistatin D ENA 78 CXCL5 GRO alpha beta gamma HB EGF D GCSF GM CSF 1 309 TCA 3 CCL1 E HGF IFN gamma IGF 1 E IL 1 beta IL 4 IL 10 F IL 1 alpha IL 2 IL 6 F IL 12 p40 IL 12 p70 I TAC CXCL11 G IL 8 CXCL8 IL 17A IP 10 CXCL10 G MCP 2 CCL8 MCP 3 MARC CCL7 MCP 4 CCL13 H Leptin LIF MCP 1 CCL2 H MMP 1 MMP 9 PECAM 1 CD31 PDGF BB PLGF RANTES CCL5 l TGF alpha TGF beta 3 Tie 1 J TGF beta 1 TIMP 1 TIMP 2 J Tie 2 uPAR VEGF A K TNF alpha TNF beta TNFSF1B Thrombopoietin TPO K VEGFR2 VEGFR3 VEGF D X Array Data Analysis Tool The RayBio Analysis Tools are array specific Excel based program that perform sophisticated data analysis on the raw numerical data extracted from the array scan see below for description The Analysis Tool specific for this array is catalog number GSH ANG 2000 SW Key features e Simplicity Easy to operate and requires no professional training With a simple copy and paste process the cytokine expression levels are determined per sample e Outlier Marking
5. amp Removing The software can automatically mark and remove the outlier spots for more accurate data analysis e Normalization The program allows for intra and inter slide normalization for large numbers of samples e Two Positive Controls The program utilizes the two positive controls in each array for normalization e User Intervention The program allows for user manual handling of outliers and other analytical data e Analyze Multiple Slide The data for multiple slides can be inputted for easy slide to slide comparison XI Troubleshooting Guide Problem Cause Recommendation Inadequate detection Increase laser power and PMT parameters Inadequate reagent Check pipettes and ensure correct volumes or improper dilution preparation E rr Increase incubation time or change sample hort in Weak Signal Sonne Hbavlonuniie incubation step to overnight Too low protein Lessen dilution or do not dilute sample concentration in sample Concentrate sample if necessary inbraparsiorida SEE Store kit as suggested temperature Don t BoR 9 freeze thaw the slide Bubble formed during Decrease amount of rocking shaking during incubations check for bubble formation and incubation remove bubbles E Arrays are not Uneven signal completed covered by Completely cover arrays with solution for all required steps reagent Paacankevaborniion Cover the incubation chamber with 9 p adhesive film during incubation Overexposur
6. ased Analysis Tool software This array as well as all catalog numbers beginning with GS differ from the classic G Series Arrays in a few important ways First each capture antibody is printed in quadruplicate instead of duplicate delivering higher precision Secondly this array features the same antibody panels used in our Quantibody Arrays allowing a seamless transition to our quantitative multiplex assay platform Lastly all 16 wells are spotted as sub arrays delivering easy handling of 16 samples simultaneously while consuming low sample volumes 10 100 ul per array ll How It Works Sample yyy gt ee Antibody array support i Y Y glass slide Incubation of sample a af 1 1 2 hours Incubation with biotinylated antibody cocktail 7 Y Y 1 2 hours ai Incubation with Cy3 equivalent dye labeled streptavidin 1 hour Scan and perform data extraction amp analysis IV Materials Provided This product is a combination of multiple arrays Items 1 5 amp 6 are array specific i Catalog Component Name 1 Slide Box GR Array Cat B a Biotinylated Antibody 1 25 ul 2x 1 25 ul i Cy3 equivalent dye conjugated QA SWD Slide Washer Dryer 1 x 30 ml Tube 4 slide kits are comprised of 2 separate 2 slide kits V Storage Upon receipt all components should be stored at 20 C The kit will retain activity for up to 6 months Once thawed the glass slide antibody cocktail and dye conjugate
7. d 100 ul Sample Diluent into each well and incubate at room temperature for 30 minutes to block slides 3 Decant buffer from each well Add 100 ul of sample to each well Incubate arrays at room temperature for 1 2 hour Longer incubation time is preferable for higher signals This step may be done overnight at 4 We recommend using 50 to 100 ul of original or diluted serum plasma conditioned media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates Cover the incubation chamber with adhesive film during incubation especially if less than 70 ul of sample or reagent is used 4 Wash e Decant the samples from each well and wash 5 times 5 min each with 150 ul of 1X Wash Buffer at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20x Wash Buffer with H20 e Optional for Cell and Tissue Lysates Put the glass slide with frame into a box with 1X Wash Buffer cover the whole glass slide and frame with Wash Buffer and wash at room temperature with gentle shaking for 20 min e Decant the 1x Wash Buffer from each well wash 2 times 5 min each with 150 ul of 1X Wash Buffer Il at room temperature with gentle shaking Completely remove wash buffer in each wash step Dilute 20X Wash Buffer II with H2O Incomplete removal of the wash buffer in each wash step may cause dark spots the background signals higher than the spots C Incubation with Biotinylate
8. d Antibody Cocktail amp Wash 5 Reconstitute the detection antibody by adding 1 4 ml of Sample Diluent to the tube Spin briefly Add 80 ul of the detection antibody cocktail to each well Incubate at room temperature for 1 2 hour Longer incubation time is preferable for higher signals 7 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer and then 2 times with 150 ul of 1x Wash Buffer II at room temperature with gentle shaking Completely remove wash buffer in each wash step D Incubation with Cy3 Equivalent Dye Streptavidin amp Wash 8 After briefly spinning down add 1 4 ml of Sample Diluent to Cy3 equivalent dye conjugated streptavidin tube Mix genily Add 80 ul of Cy3 equivalent dye conjugated streptavidin to each well Cover the device with aluminum foil to avoid exposure to light or incubate in dark room Incubate at room temperature for 1 hour 10 Decant the samples from each well and wash 5 times 5 mins each with 150 ul of 1X Wash Buffer at room temperature with gentle shaking Completely remove wash buffer in each wash step E Fluorescence Detection 11 12 13 14 Disassemble the device by pushing clips outward from the slide side Carefully remove the slide from the gasket Be careful not to touch the surface of the array side Place the slide in the Slide Washer Dryer a 4 slide holder centrifuge tube add enough 1x Wash Buffer
9. d Streptavidin should be kept at 20 C All other components may be stored at 4 C The entire kit should be used within 6 months of purchase VI Additional Materials Required e Benchtop rocker or orbital rocker e Laser scanner for fluorescence detection e Aluminum foil e Distilled water e 1 5 ml Polypropylene microcentrifuge tubes VII General Considerations A Preparation of Samples e Use serum free conditioned media if possible e f serum containing conditioned media is required it is highly recommended that complete medium be used as a control since many types of sera contains cytokines e We recommend the following parameters for your samples 50 to 100 ul of original or diluted serum plasma cell culture media or other body fluid or 50 500 ug ml of protein for cell and tissue lysates If you experience high background or if the fluorescent signal intensities exceed the detection range further dilution of your sample is recommended B Handling Glass Slides e Do not touch the surface of the slides as the microarray slides are very sensitive Hold the slides by the edges only e Handle all buffers and slides with powder free gloves Handle glass slide s in clean environment The GS Series slides do not have bar codes To help distinguish one slide from another transcribe the slide serial number from the slide bag to the back of the slide with a fine point permanent marker Please write the number on the ve
10. e Lower the PMT or sigmal gain akspis Completely remove wash buffer in each wash step High Increase wash time and use more wash Insufficient wash background buffer Dust Work in clean environment rag Silence to diy Don t dry out slides during experiment XII Publications Citing This Product 1 Koob T Lim J Massee M Zabek N Rennet R Gurtner G Li W Angiogenic properties of dehydrated human amnion chorion allografts Vascular Cell 2014 6 10 doi 10 1186 2045 824X 6 10 Epub Ahead of Print therapeutic potential for soft tissue repair and regeneration Species Human Sample Type Conditioned Media More citations for this product may be available Contact techsupport raybiotech com Note The citations listed above are for the Quantibody product line which is the same as the GS Series but include protein standards for quantitation Also The citations listed above are for the use of this combination array Citations for the individual arrays can be found in the individual array manuals XIII Experiment Record Form Date File Name Laser Power PMTL ooo Weli No o a pe EM nm o pele de e Ie de lls 2 E E 2 XIV How to Choose a GS Series Array Species based selection Human GSH Mouse GSM Rat GSR Bovine GSB Canine GSC Equine GSE Feline GSF Ovine GSO Primates GSN Porcine GSP Rabbit GSL Function based selection Adhesion M
11. olecule Angiogenesis Arrays Bone Metabolism Chemokine Arrays Arrays Arrays El Biomarker Custom Arrays Cytokine Arrays Growth Factor Arrays UA IL 1 Family Arrays midi nesponce Inflammation Arrays Arrays Arrays Interleukin Arrays Isotyping Arrays MMP Arrays Obesity Arrays Ophthalmic Arrays Mo Disgagg Receptor Arrays Th1 Th2 Th17 Arrays Cytokine Number based selection Arrays are available in the GS Series amp Quantibody platform to detect 660 human 200 mouse or 67 rat proteins GLP Compliant testing services are also available This product is for research use only 2015 RayBiotech Inc
12. ry bottom edge of the slide taking care to avoid writing on the array well areas C Incubation e Completely cover array area with sample or buffer during incubation e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rocking or rotation e Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or lt 70 ul of sample or reagent is used e Several incubation steps such as step 6 blocking step 7 sample incubation step 10 detection antibody incubation or step 13 Cy3 equivalent dye streptavidin incubation may be done overnight at 4 C Please make sure to cover the incubation chamber tightly to prevent evaporation VIII Protocol Note This product contains sets of reagents for different arrays Always ensure you are using the proper glass slide and biotinylated antibody cocktail for the correct corresponding array The following procedure is for processing any one of the arrays in the kit A Completely Air Dry The Glass Slide 1 Take out the glass slide from the box and let it equilibrate to room temperature inside the sealed plastic bag for 20 30 minutes Remove slide from the plastic bag peel off the cover film and let it air dry for another 1 2 hours Incomplete drying of slides before use may cause the formation of comet tails thin directional smearing of antibody spots B Blocking amp Incubation 2 Ad
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