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1. 16 C Background Subtraction ccc 17 D Normalization of Array Data 17 E Threshold of Significance 18 VI Antibody Array Map cccccceeceescsssesssssseesnnsnsnnnnen 19 Vil Troubleshooting Guide cssssssessssseeeeeseee 20 VIII Selected References ccc 22 RayBio Cytokine Antibody Arrays are patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Rat Cytokine Antibody Array G Series 1 Introduction New techniques such as cDNA microarrays have enabled us to analyze global gene expression However almost all cell functions are executed by proteins which cannot be studied simply through DNA and RNA techniques Experimental analysis clearly shows disparity can exist between the relative expression levels of mRNA and their corresponding proteins Therefore analysis of the proteomic profile is critical The conventional approach to analyzing multiple protein expression levels has been to use 2 D SDS PAGE coupled with mass spectrometry However these methods are slow expensive labor intensive and require specialized equipment Thus effective study of multiple protein expression levels can be complicated costly and time consuming Moreover these traditional methods of proteomics are not sensitive enough to detect most cytokines typically at pg ml concentrations Cytokines broadly defined as secreted cell cell signaling proteins distinct from classic
2. light or incubate in dark room Incubate at RT for 2 hours with gentle rocking or shaking 15 Remove aluminum foil and adhesive film Carefully aspirate the Streptavidin Fluor reagent Wash as described in Step 7 above first with Wash Buffer then with Wash Buffer II making sure to completely remove buffer between washes and after final wash 16 Remove the glass chip from the frame assembly Place the whole chip in 30 ml centrifuge tube provided or slide staining jar Add enough Wash Buffer to cover the whole slide about 20 ml and gently rock or shake at RT for 10 min 17 Decant buffer and repeat wash as described in Step 16 1 x 10 min with Wash Buffer 18 Decant buffer and repeat wash as described in Step 16 but this time using Wash Buffer II for only 2 3 minutes 19 Decant buffer remove the glass chip from the tube then gently rinse the slide with de ionized H20 using a plastic wash bottle 20 Remove water droplets by applying suction gently with a pipette tip NOTE Be careful not to touch the array portions of the slide with your pipette tip only touch the sides of the slide C Obtaining Fluorescent Signal Intensities 21 Allow glass chip to dry in a laminar flow hood for 20 minutes or until slide is completely dry Place chip under an aluminum foil tent to protect it from light Make sure the slides are absolutely dry before scanning or storage 22 You may proceed immediately to scanning Step 23 o
3. Use local background correction also median value Exclude obvious outlier data in its calculations Scan all slides at same PMT 18 RayBio Rat Cytokine Antibody Array G Series 1 Vi RayBio Rat Cytokine Antibody Array G Series 1 Map Detects 19 Rat Cytokines in one experiment A B C D E F G H 1 POS1 POS2 POS3 NEG NEG CINC2 CINC3 CNTF CX3CL1 2 POS1 POS2 POS3 NEG NEG CINC2 CINC3 CNTF CX3CL1 3 CSF2 IFN y IL 4a IL 16 IL 4 IL 6 IL 10 LIX Leptin 4 CSF2 IFN y IL 4a IL 16 IL 4 IL 6 IL 10 LIX Leptin 5 MCP1 MIP 3a B NGF TIMP1 TNF a VEGF A NEG NEG POS4 6 MCP1 MIP 3a B NGF TIMP1 TNF a VEGF A NEG NEG POS4 Notes on Array Map e CINC2 CXCL3 human GRO y homolog CINC3 CXCL2 human GRO B homolog CX3CL1 Fractalkine CSF2 GM CSF LIX CXCL5 e VEGF A detects VEGF 165 aa and VEGF 121 aa RayBio Rat Cytokine Antibody Array G Series 1 19 VII Troubleshooting guide Problem Cause Recommendation No signal for any spots including Positive Controls Global detection failure Adjust scanner settings or re assemble chip into holder wash slide 2 x 5 min with 150 ul Wash Buffer II and repeat Steps 12 21 Similar signal intensities for POS1 2 3 Improper laser power and or PMT setting Repeat scan using higher and or lower laser power or PMT settings High background signals Incomplete washes Care
4. glass chip assembly into the container Add enough 1X Wash Buffer to submerge the entire glass chip with frame intact approx 30 50 ml and remove all bubbles in wells Wash 10 min at RT with gentle rocking or shaking 9 Remove assembled glass chip from container and invert to decant liquid Decant buffer from container and replenish with 1X Wash Buffer Submerge the entire glass chip assembly and wash 10 min at RT with gentle rocking or shaking 10 Remove assembled glass chip from container and invert to decant liquid Decant buffer from container and repeat Steps 8 amp 9 with Wash Buffer Il 11 Remove assembled glass chip from container and invert to decant liquid then carefully aspirate wash buffer from wells touching only the corners with your pipette tip 12 Add 70 ul of 1X Biotin conjugated Anti Cytokines to each sub array Cover incubation chamber with Adhesive film included in kit Incubate at RT for 2 hours with gentle rocking or shaking 13 Carefully aspirate all of the Biotin conjugated Anti Cytokine reagent Wash as described in Step 7 above first with Wash Buffer then with Wash Buffer Il making sure to completely remove buffer between washes and after final wash 14 Add 70 ul of 1X Streptavidin Fluor to each sub array Cover the incubation chamber with Adhesive film included in kit then cover entire assembly with aluminum foil to avoid exposure to 13 RayBio Rat Cytokine Antibody Array G Series 1
5. of this easy to use array format see Section VIII RayBio Rat Cytokine Antibody Array G Series 1 1 Tang X Marciano DL Leeman SE Amar S LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT6 B with effects on multiple cytokines PNAS 2005 102 14 5132 5137 2 Xu Y Kulkosky J Acheampong E et al HIV 1 mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced encephalopathy PNAS 2004 101 18 7070 7075 3 El Hage N Gurwell JA Singh IN et al Synergistic increases in intracellular Ca 2 and the release of MCP 1 RANTES and IL 6 by astrocytes treated with opiates and HIV 1 Tat G ia 2005 50 2 91 106 4 OhHS Moharita A Potian JG et al Bone Marrow Stroma Influences Transforming Growth Factor 8 Production in Breast Cancer Cells to Regulate c myc Activation of the Preprotachykinin I Gene in Breast Cancer Cells Cancer Res 2004 64 6327 6336 5 Osorio Y Ghiasi H Recombinant Herpes Simplex Virus Type 1 HSV 1 Codelivering Interleukin 12p35 as a Molecular Adjuvant Enhances the Protective Immune Response against Ocular HSV 1 Challenge J Virol 2005 79 6 3297 3308 6 Mullick A Elias M Picard S Bourget L et al Dysregulated Inflammatory Response to Candida albicans in a C5 Deficient Mouse Strain nfect Immunity 2004 72 10 5868 5876 7 Maruvada P Wang W Wagner PD Srivastava S Biomarkers in molecular medicine cancer detection and dia
6. ul of each sample to each sub array Cover the incubation chamber with Adhesive film included in kit Incubate arrays with sample at RT for 2 hours Dilute sample using 1X Blocking Buffer if necessary RayBio Rat Cytokine Antibody Array G Series 1 6 Remove adhesive film and carefully aspirate samples from sub arrays touching only the corners with your pipette tip NOTE Try to prevent solution from flowing into neighboring wells Instructions for incubation chamber assembly G Series and Quantibody Arrays Incubation chamber gt Gasket normally attached to chamber Protective Cover can a is be discarded Carefully place slide at bottom of the chamber as shown The slide will adhere somewhat to the bottom Warning the slide is fragile so do not apply more than gentle force to the apparatus While gently holding chamber and slide place side on chamber as shown beginning with bottom flap first Then press the top of the side into grove on chamber and then apply even gentle pressure from one end to the other Repeat this procedure with the other side 12 RayBio Rat Cytokine Antibody Array G Series 1 7 Wash 3 x 2 min with 150 ul 1X Wash Buffer at RT Be sure to completely remove sample and Wash Buffer each time and use fresh buffer for each wash Decant final wash solution before proceeding to next step 8 Obtain a clean container eg pipette tip box or slide staining jar and place
7. Blocking Buffer to a final volume of 1500 ul ie 1 5 ml Wrap tube containing Streptavidin Fluor with aluminum foil This working dilution can be stored for 3 5 days at 4 C RayBio Rat Cytokine Antibody Array G Series 1 10 B Blocking and Incubations NOTE Please carefully read Section III of this manual before proceeding NOTE Prepare all reagents immediately prior to use as described above Section IV A and before proceeding 1 Remove the package containing the glass chip assembly from the freezer Place unopened package on the benchtop and allow the glass chip assembly to equilibrate to room temperature RT approx 15 min Open package remove the glass chip assembly and place in laminar flow hood to dry for 1 2 hours NOTE Be sure glass chip is completely dry before proceeding 2 If necessary assemble the glass chip into incubation chamber and frame as shown on page 12 Note if you slide is already assembled you can proceed directly to Step 3 3 Add 100 ul 1 X Blocking Buffer into each well and incubate at RT for 30 min to block slides NOTE Only add reagents or samples to wells printed with antibodies see diagram on page 5 4 Decant Blocking Buffer then aspirate remaining liquid from each well NOTE To aspirate liquid samples or reagents from wells gently place the pipette tip only in the corners of the well Do not scrape the pipette tip across the surface of the chip 5 Add 50 to 100
8. P Antibody Arrays o Obesity Antibody Arrays Quantibody Multiplex ELISA Arrays RayBio L Series Biotin Label based Antibody Arrays RayBio E Series Competition based Antibody Arrays RayBio Phosphorylation Antibody Arrays o Receptor Tyrosine Kinases o EGFR and ErbB family site specific phosphorylation Over 1 300 different ELISA kits EIA Competitive ELISA kits Cell based Phosphorylation Assay Over 20 000 different antibodies Recombinant proteins Peptide Recombinant antibodies TABLE OF CONTENTS l WMO GI CUO eneee 1 Il Product INFOrMAtION cece teteesccsseeteeteeeecnsneneeeeeeennenneees 5 A Storage Recommendations ce ee 5 B RayBio G Series Glass Chip Layout 6 C Materials Provided 0 ccc 6 D Additional Materials Required 000c 6 E How TE Works ractcccsecctccciescarshedasccscttsctatstccetctcvenvarctnonsecteaie 7 Ill Helpful Tips and General Considerations 7 A Preparation and Storage of Samples 7 B Handling Glass Chip c ccceeeeeeeeeeieee 9 C Incubations and WaSh6 S c cece 9 D Data Extraction Tips 9 IV ProtOCO eerren anaha a iE 10 A Preparation and Storage of Reagents 10 B Blocking and Incubations c c cee 11 C Fluorescence Detection 14 V Interpretation of Results 15 A Explanation of Control Spots 15 B Typical Results using G Series Arrays
9. RayBio Rat Cytokine Antibody Array 1 G Series Patent Pending Technology User Manual Revised June 5 2014 RayBio Rat Cytokine Antibody Array G Series 1 Cat AAR CYT G1 4 RayBio Rat Cytokine Antibody Array G Series 1 Cat AAR CYT G1 8 RayBio Rat Cytokine Antibody Array G Series Testing Service Cat AAR SERV G Please read manual carefully before starting experiment Ca RayBiotech Inc Hi the protein array pioneer company We provide you with excellent Protein Array systems and services Tel Toll Free 1 888 494 8555 or 1 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com Email info raybiotech com RayBiotech Inc the Protein Array Pioneer Company strives to research and develop new products to meet demands of the biomedical community RayBiotech s patent pending technology allows detection of up to 1 000 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable reproducible and cost effective Our product offerings include wo o N ATOE oS Protein antigen Arrays RayBio Cytokine Antibody Arrays C Series Membrane chemiluminescence detection G Series Glass chip fluorescence detection Pathway and disease focused antibody arrays o Angiogenesis Antibody Arrays o Apoptosis Antibody Arrays o Atherosclerosis Antibody Arrays o Chemokine Antibody Arrays o Growth Factor Antibody Arrays o Inflammation Antibody Arrays o MM
10. aration and Storage of Reagents NOTE During this protocol prepare reagents immediately prior to use and keep working dilutions of all reagents on ice at all times i Blocking Buffer is supplied at 1X concentration No dilution is required Wash Buffers and II are supplied at 20X concentration a b For each glass chip 4 or 8 sub arrays chip dilute 6 ml of 20X concentrate with deionized H20 to a final volume of 120 ml each of Wash Buffer amp Wash Buffer Il Wash buffer reagents at working dilution 1X can be stored at 4 C for up to 1 month Stock solutions at 20X can be stored 4 C for up to 3 months Biotin conjugated Anti Cytokines are supplied at high concentration in a small liquid bead typically 2 5 ul Spin down the tube prior to reconstitution as the concentrated liquid bead may have moved to the top of the tube during handling Prepare stock reagent by adding 300 ul 1X Blocking Buffer to Biotin Conjugated Anti Cytokines Mix well 1X Biotin Conjugated Anti Cytokines may be stored for 2 3 days at 4 C Streptavidin Fluor is supplied at 1500x concentration Mix the tube containing 1500X Streptavidin Fluor well before use as precipitants may form during storage Add 100 ul of 1X Blocking Buffer to tube containing 1500X Streptavidin Fluor Mix well Quantitatively transfer all of Streptavidin Fluor reagent from the original tube to a larger one and dilute with 1X
11. ed Serum amp Plasma 5 fold to 10 fold dilution Most other Body Fluids Neat or 2 fold to 5 fold dilution Cell and Tissue Lysates Minimum 5 fold to 10 fold to equal concentrations of total protein in each lysate sample o You must determine the total protein concentration of each lysate homogenate We recommend using the BCA method available from Pierce it is insensitive to detergents commonly found in lysis buffers o Minimum Recommended Dilution of Lysates prior to sample incubation 5 fold to 10 fold with 1X Blocking Buffer Dilute all lysate samples to the same final concentration of total lysate protein in 1X Blocking Buffer to 100 ul final volume o To start we recommend using 10 100 ug of total protein in 100 pl of 1X Blocking Buffer final volume per sub array o Optimal amounts of total lysate protein may range from 5 500 ug per sub array Based upon background and spots intensities you may increase or decrease the amount of protein used in subsequent experiments e Other Liquid Sample Types Most often Neat or 2 fold to 5 fold However optimal dilutions should be determined empirically 3 Sample Preparation For tips on sample preparation please visit our Website http www raybiotech com Tech Support SampleTips pdf RayBio Rat Cytokine Antibody Array G Series 1 B Handling Glass Chips Do not remove glass chip from assembly until Step 16 Hold the slides by edges only do not touch the surface Handle a
12. ely Negative Control NEG spots are a protein containing buffer used to dilute antibodies printed on the array Their signal intensities represent non specific binding of Biotin conjugated anti Cytokines and or Streptavidin Fluor Negative control signal intensities are usually very close to background signals in each sub array 15 RayBio Rat Cytokine Antibody Array G Series 1 B Typical results from RayBio G Series Antibody Arrays The following figure shows typical results obtained using RayBio Antibody Array G Series Arrays The images were captured using a GenePix 4000B scanner gl Array VI Array VII Array VIII Patient Serum 1 Patient Serum 2 Patient Serum 3 Negative Control In this example sera from several patients were incubated with Human Cytokine Arrays 6 7 amp 8 sold together as Human Cytokine Array G Series 2000 AAH CYT G2000 4 or AAH CTY G2000 8 and processed using this standard protocol The 6 strong signals of the Positive Control spots in the upper left corner are useful for proper orientation of the array image If scanned using optimal scan settings 3 distinct Positive Control signal intensities will be seen POS1 gt POS2 gt POS3 If all of these signals are of similar intensity try increasing or decreasing laser power and or signal gain settings Once you have obtained fluorescence intensity data you should subtract the background and normalize to the Positive Control
13. entration and or incubate wi sample O N at 4 C Increase concentration of and or length of incubation with Biotin conjugated Anti Cytokine add l large volume wash following Biotin Ab incubation Review proper storage conditions for kit components RayBio Rat Cytokine Antibody Array G Series 1 21 Ill Selected References Citing RayBio G Series Arrays 1 Light M Minor KH DeWitt P Jasper KH Davies SJA Multiplex array proteomics detects increase MMP 8 in CSF after spinal cord injury J Neuroinflamm 2012 9 122 doi 10 1186 1742 2094 9 122 Fong CC Wei F Chen Y Yu WK Koon CM et al Danshen Gegen decoction exerts proliferative effect on rat cardiac myoblasts H9c2 via MAPK and insulin pathways J Ethnopharmacol 2011 138 1 60 66 Cohen TS Lawrence GG Margulies SS Cultured alveolar epithelial cells from septic rats mimic n vivo septic lung PLoS One 2010 5 6 11322 doi 10 1371 journal pone 0011322 Cohen TS Bucki R Byfield FJ Ciccarelli NJ Rosenberg B et al Therapeutic potential of plasma gelsolin administration in a rat model of sepsis Cytokine 2011 54 3 235 238 Chopy D Pthlichet J Lafage M M gret F et al Ambivalent role of the innate immune response in Rabies virus pathogenesis J Viro 2011 85 13 6657 6668 Rajanbabu V Pan C Y Lee S C Lin W J et al Tilapia Hepcidin 2 3 peptide modulates lipopolysaccharide induced cytokines and inhibits tumor necrosis factor a thr
14. fully follow wash protocols and or increase wash times Sample concentration is too high Repeat using lower sample concentration Fluor and or Anti Cytokines are too concentrated Review protocol for dilution of reagents Uneven background and or missing spots Bubbles present on chip during incubations Be sure to completely remove all bubbles from chip surface Evaporation during incubation steps Cover chamber assembly during washes and incubations Pooling precipitation of sample or reagent Incomplete washes Cover chamber assembly and use a rocker or shaker during washes and incubations carefully follow wash protocols Sample is too concentrated Repeat experiment using more dilute sample Randomly scattered high intensity spots Dust or other particulates Dry slides in laminar flow hood and or use clean containers and powder free gloves RayBio Rat Cytokine Antibody Array G Series 1 20 Weak or no signals antigen specific pots Low Background Sample is too dilute Repeat experiment using higher sample concentration Improper dilution of Anti Cytokines or Streptavidin Fluor Re assemble chip into holder wash 2 x 5 min with 150 ul Wash Buffer II and repeat Steps 12 21 Spin down reagents before diluting and mix well Other Tips Rescan at higher laser power or signal gain setting Repeat using higher sample conc
15. gnosis Biotechniques 2005 38 S9 S15 8 Bartek J Hodny Z Lukas J Cytokine loops driving senescence Nature Cell Biol 2008 10 887 889 9 Minami K Yanagawa Y Iwabuchi K et al Negative feedback regulation of T helper type 1 Th1 Th2 cytokine balance via dendritic cell and natural killer T cell interactions Blood 2005 106 1685 1693 10 Ozaki K Leonard WJ Cytokine and Cytokine Receptor Pleiotrophy and Redundancy J Biol Chem 2002 227 29355 29358 11 Ragab AA Nalepka JL Bi Y Greenfield EM Cytokines synergistically induce osteoclast differentiation support by immortalized or normal calvarial cells Am J Physiol Cell Physiol 2002 283 3 C679 C687 12 Devalaraja MN Richmond A Multiple chemotactic factors fine control or redundancy 7rends Pharmacol Sci 1999 20 4 151 156 13 Heaney ML Golde DE Soluble Cytokine Receptors Blood 1996 87 3 847 857 RayBio Rat Cytokine Antibody Array G Series 1 Il Product Information A Storage Recommendations For best results we recommend storing the entire kit at 20 C or 80 C upon arrival and using the kit within 6 months of receipt RayBiotech warranties this product for 6 months if stored in this manner Once thawed store glass chips and 1X Blocking Buffer at 20 C or 80 C and all other component at 4 C After thawing the entire kit should be used within 3 months RayBio Antibody Array kits are robust and will retain full activity even if acciden
16. hormones or neurotransmitters play important roles in inflammation innate immunity apoptosis angiogenesis cell growth and differentiation They are involved in most diseases including cancer obesity and inflammatory and cardiac diseases Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool to study cytokines Regulation of cellular processes by cytokines is a complex dynamic process often involving multiple proteins Positive and negative feedback loops pleiotropic effects and redundant functions spatial and temporal expression of or synergistic interactions between multiple cytokines even regulation via release of soluble forms of membrane bound receptors all are common mechanisms modulating the effects of cytokine signaling As such unraveling the role of individual cytokines in physiologic or pathologic processes generally requires consideration and detection of multiple cytokines rather than of a single cytokine RayBio Rat Cytokine Antibody Array G Series 1 RayBio G Series Cytokine Antibody Arrays have several advantages over detection of cytokines using single target ELISA 1 More Data Less Sample Antibody arrays provide high content screening using about the same sample volume as for ELISA 2 Global View _of Cytokine Expression Antibody array screening improves the chances for discovering key factors disease mechanisms or biomarkers related to cytokine signaling 3 Greater Sen
17. ize signal intensities to the Positive Controls To order the Analysis Tool please contact us at 1 770 729 2992 or info raybiotech com for more information 17 RayBio Rat Cytokine Antibody Array G Series 1 E Threshold of significant difference in expression After subtracting background signals and normalization to Positive controls comparison of signal intensities for antigen specific antibody spots between and among array images can be used to determine relative differences in expression levels of each analyte ie protein detected between samples or groups Any 21 5 fold increase or lt 0 65 fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression provided that both sets of signals are well above background Mean background 2 standard deviations accuracy 95 NOTE In the absence of an external standard curve for each analyte there is no means of assessing absolute or relative concentrations of different analytes in the same sample using immunoassays If you wish to obtain quantitative data le concentrations of the various analytes in your samples try using our Quantibody Multiplex ELISA arrays instead Data Extraction Tips e Ignore any comet tails Define the area for signal capture for all spots as 110 120 micron diameter using the same area for every spot Use median signal value not the total or the mean
18. ker Aluminum foil Gene microarray scanner or similar laser fluorescence scanner see pages 9 amp 15 RayBio Rat Cytokine Antibody Array G Series 1 D How It Works Array support YYYYY z ro Samples 4 Incubation of Sample 1 2 hrs with arrayed antibody OIN is ma AX Biotin Ab Al AK Incubation with yY Biotinylated Ab idii Labeled ooo streptavidin i Incubation with 1 hrs labeled Streptavidin Zot 7 cea of i signals Data analysis and graph Ill Helpful Tips and General Considerations A Preparation and Storage of Samples 1 General Considerations e Freeze samples as soon as possible after collection e Avoid multiple freeze thaw cycles If possible sub aliquot your samples prior to initial storage e Spin samples hard 5 10 minutes at 10K to 15K RPM immediately prior to incubation of samples with array e Optimal sample concentrations may need to be determined empirically based on the signal intensities of spots and background signals obtained RayBio Rat Cytokine Antibody Array G Series 1 e Most samples will not need to be concentrated If concentration is required we recommend using a spin column concentrator with a chilled centrifuge 2 Recommended Sample Volumes and Dilution Factors NOTE All sample dilutions should be made using 1X Blocking Buffer For all sample types final sample volume 50 700 ul per sub array Cell Cultured Media Neat no dilution need
19. ll buffers and slides with powder free gloves Dry glass chip completely before proceeding to Step 3 Handle and dry glass chip in clean environment Avoid breaking glass chip when removing the chamber assembly C Incubations and Washes Cover incubation chamber with adhesive film included in kit to prevent evaporation particularly during incubation or wash steps gt 2 h or with liquid volumes lt 100 ul per well Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle s Wash steps in Wash Buffer II and all incubation steps may be performed overnight at 4 C o Overnight sample incubations are the most effective at increasing sample spot intensities Avoid cross contamination of samples to neighboring wells To remove Wash Buffers and other reagents from chamber wells you may invert the Glass Chip Assembly to decant and aspirate the remaining liquid In Wash Steps 6 12 and 15 you may gently flush wells several times using a wash bottle filled with Wash Buffer I D Scanning and Data Extraction Tips For tips on scanning and data extraction please visit our Website http www raybiotech com Tech Support Scanning Tips pdf For a list of recommended scanners please visit our Website http www raybiotech com Tech Support Laser Scanners for Glass Slide Arrays pdf See also page 18 of this manual RayBio Rat Cytokine Antibody Array G Series 1 IV Protocol A Prep
20. ngton RD Cytokine antibody array analysis in brain and periphery of scrapie infected Tg338 mice Compar Immunol Microbiol Infect Dis 2011 34 5 387 397 13 Nolting T Lindecke A Koutsilie E Maschke M et al Measurement of soluble inflammatory mediators in cerebrospinal fluid of human immunodeficiency virus positive patients at distinct stages of infection by solid phase protein array J Neruovirol 2009 15 5 6 390 400 14 Pannebaker C Chandler HL Nichols JJ Tear proteomics in keratoconus Mol Vision 2010 16 1949 1957 23 RayBio Rat Cytokine Antibody Array G Series 1 Customized RayBio Cytokine Antibody Arrays Select your Rat cytokines of interest from the following list and we will produce the customized array for you For more information please visit our website www raybiotech com B7 2 GM CSF IL 6 Prolactin R Beta NGF ICAM 1 IL 10 RAGE CINC1 IFN gamma IL 13 Thymus CK 1 CINC2 IL 1 alpha Leptin TIMP 1 CINC3 IL 1 beta LIX TNF alpha CNTF IL1 R6 L Selectin VEGF A Fas Ligand IL 2 MCP 1 Fractalkine IL 4 PDGF AA RayBio Rat Cytokine Antibody Array G Series 1 24 Testing Services RayBiotech offers full testing services using any of our Array ELISA or EIA products including customized products Just send your samples and we will send you the results Custom Services Customized Antibody and Protein Arrays Customized Phosphorylation Arrays Peptide synthesis Peptide arrays Recombinant protein and a
21. ntibody production ELISA EIA Assay development Biostatistical amp Bioinformatic Analysis 0 Peptoid Synthesis amp Library Screening a PA oo Pen Technology Transfer Program Have you developed technologies or reagents of interest to the scientific and research community RayBiotech can help you commercialize your technologies reagents and your dreams 25 RayBio Rat Cytokine Antibody Array G Series 1 RayBio Cytokine Antibody Arrays are patent pending technology developed by RayBiotech This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for 6 months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price RayBio is a registered trademark of RayBiotech Inc HiLyte Plus is a trademark of Anaspec Inc InnoScan is a registered trademark of Innopsys Inc This product is for research use only 2012 RayBiotech Inc RayBio Rat Cytokine Antibody Array G Series 1 26
22. ough cyclooxygenase 2 and phosphodiesterase 4D J Biol Chem 2010 285 30577 30586 Duncan JA Gao X Huang MT H O Connor BP Thomas CE et al Neisseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling Activities of the NLRP3 and ASC Containing Inflammasome J mmunol 2009 182 6460 6469 Pukstadad BS Ryana L Floa TH JStenvika J et al Non healing is associated with persistent stimulation of the innate 22 RayBio Rat Cytokine Antibody Array G Series 1 immune response in chronic venous leg ulcers J Dermatol Sa 2009 59 2 115 122 9 Park JE Tan HS Datta A Lai RC et al Hypoxic Tumor Cell Modulates Its Microenvironment to Enhance Angiogenic and Metastatic Potential by Secretion of Proteins and Exosomes Mol Cell Proteom 201 9 1085 1099 10 Streblow DN Dumortier J AMoses AV Orloff SL Nelson JA Mechanisms of Cytomegalovirus Accelerated Vascular Disease Induction of Paracrine Factors That Promote Angiogenesis and Wound Healing Shenk TE Stinski MF eds Current Topics in Microbiology and Immunology Human Cytomegalovirus Berlin Heidelberg Germany Springer 2008 325 397 415 11 Long C Hosseinkhani MR Wang Y Sriramarao P Walcheck B ADAM17 activation in circulating neutrophils following bacterial challenge impairs their recruitment J Leuk Biol 2012 published online before print doi 10 1189 jlb 0312112 12 Newsom DM Liggitt HD O Rourke K Zhuang D Schneider DA Harri
23. r you may scan at a later time You may store the slides at RT indefinitely provided they are protected from strong light RayBio Rat Cytokine Antibody Array G Series 1 Note Unlike most Cy3 fluors the HiLyte Plus Fluor 555 used in this kit is very stable at RT and resistant to photobleaching on completed glass chips However please protect glass chips from strong light and temperatures above RT 23 Scan the glass chip with a laser scanner such as Innopsys InnoScan using cy3 or green channel excitation frequency 532 nm For tips on scanning visit our Website http www raybiotech com Tech Support Scanning Tips pdf NOTE If you do not have a laser scanner for a nominal fee you can send your slide to us for scanning and data extraction using Innopsys InnoScan and we will return the results to you Using using alternate protocols RayBio G Series arrays are also compatible with Li Cor s Odyssey and other microarray scanners V Interpretation of Results A Explanation of Controls Spots Positive Controls POS1 POS2 POS3 are equal amounts of biotinylated IgGs printed directly onto the array All other variables being equal the Positive Control intensities will be the same for each sub array This allows for normalization based upon the relative fluorescence signal responses to a known control much as housekeeping genes or proteins are used to normalize results in PCR or Western blots respectiv
24. signals before proceeding to analysis RayBio Rat Cytokine Antibody Array G Series 1 C Background Subtraction Most laser fluorescence scanner software have an option to automatically measure the local background around each spot As with spot signal intensities we recommend using MEDIAN background signals If your resulting fluorescence signal intensity reports do not include these values eg a column labeled as MED532 B532 you may need to subtract the background manually or change the default settings on your scanner s data report menu D Normalization of Array Data To normalize signal intensity data one sub array is defined as reference to which the other arrays are normalized This choice can be arbitrary For example in our Analysis Tool Software the array represented by data entered in the left most column each worksheet is the default reference array You can calculate the normalized values as follows X Ny X y P1 P y Where P1 mean signal intensity of POS spots on reference array P y mean signal intensity of POS spots on Array y X y mean signal intensity for spot X on Array y X Ny normalized signal intensity for spot X on Array y The RayBio Analysis Tool software is available for use with data obtained using RayBio G Series Arrays You can copy and paste your signal intensity data with and without background into the Analysis Tool and it will automatically normal
25. sitivity As little as 4 pg ml of MCP 1 can be detected using the G Series array format In contrast our similar MCP 1 ELISA assay has a sensitivity of 40 pg ml of MCP 1 4 Increased Range of Detection ELISA assays typically detect a concentration range of 100 to 1000 fold however RayBiotech arrays can detect IL 2 at concentrations of 25 to 250 000 pg ml a range of 10 000 fold 5 Better Precision As determined by densitometry the inter array Coefficient of Variation CV of spot signal intensities is 5 10 comparing favorably with ELISA testing CV 10 15 The RayBio G Series Cytokine Antibody Array is a glass chip that is a highly sensitive approach to simultaneously detect multiple cytokine expression levels from diverse sample types The experimental procedure is simple and can be performed in any laboratory The signals from G Series arrays are detected using a laser scanner Larger multi array G Series Human Cytokine Antibody Array Kits such as the G1000 can detect hundreds of cytokines in a single experiment For example the Human G1000 arrays can detect up to 120 cytokines the Human G2000 arrays can detect up to 174 cytokines and the Human G4000 can detect up to 274 cytokines RayBiotech The Protein Array Pioneer Company introduced the first protein arrays to the market in 2001 and continues to lead in the development of innovative protein array technologies For a list of publications demonstrating the usefulness
26. tally stored at room temperature RT for up to 24 hours B RayBio G Series Glass Chip Layout BO ane Antibody pes Be rray BO E EE a Blank gt _ Blank _ L O O O O O O Barcode I IIj Barcode II IIjJ 4 arrays in one glass chip 8 arrays in one glass chip RayBio Rat Cytokine Antibody Array G Series 1 B Materials Provided AAR AAR o CYT G1 CYT G1 Item Description 4 8 1 chip with 1 chip with Roei R Ra Onna 4Sub 8 Sub Microarray Glass Chip 4 z arrays arrays 3002 Biotin Conjugated Anti Cytokines 1ea 2ea 1 500X HiLyte Plus 555 oa Streptavidin Fluort wee ee 0103004 B 1X Blocking Buffer 10 ml 20 ml 0103004 W 20X Wash Buffer 30 ml 30 ml 0103004 W 20X Wash Buffer II 30 ml 30 ml 0103004 L 2X Cell Lysis Buffer optional 10 ml 10 ml Other Kit Components Manual Adhesive Plastic Strips 30 ml Centrifuge Tube Kit contains 1 pre assembled glass chip with either 4 or 8 printed sub arrays per chip in sealed plastic envelope NOTE In some cases 2 chips x 4 sub arrays chip may be substituted in kits containing 8 sub arrays t This fluor is patent pending technology from Anaspec Inc Wash Buffers are sold as sets C Additional Materials Required Small plastic boxes or containers Pipettors pipette tips and other common lab consumables Orbital shaker or oscillating roc

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