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1. analysis tool software ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Peptide Protein arrays EIA OC Sl ya re RayBiotech also provides excellent custom service 1 Antibody arrays RayBio Rat Cytokine Antibody Array G series 16 Protein arrays Peptide synthesis Production of recombinant protein and antibody Peptide arrays Phosphorylation arrays ELISA EIA 907 aie Just simply send your samples and we will do the assay for you Technology transfer program Have you developed technologies or reagents interested to the scientific and research community RayBiotech can help you commercialize your technologies reagents and dream RayBio Rat Cytokine Antibody Array G series 17 VI Troubleshooting guide Problem Weak signal Uneven signal Cause Recommendation Inadequate detection Check laser power and PMT parameters Inadequate reagent volumes Check pipetters and or improper dilution ensure correct preparation f ee Ensure sufficient incubation incubation step to ovemight Too low protein concentration Don t nuke too low dilution in sample Or concentrate sample Improper storage of kit Store kit at suggested temperature Excess of biotinylated antibodies Make sure correct amount of antibodies Excess of streptavidin Make sure correct amount of streptavidin Inadequate detection Check laser power And PMT parameters dust Work in clean environmen2. 2007 20 665 678 Fu Y S LinY Y Chou S C Tsai T H et al Tetramethylpyrazine inhibits activities of glioma cells and glutamate neuro excitotoxicity Potential therapeutic application for treatment of gliomas Neuro Oncol 2008 10 2 139 152 RayBio Rat Cytokine Antibody Array G series 20 Note RayBio is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology 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 three 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 HiLytePlus is a trademark of AnaSpec Inc This product is for research use only 2009 RayBiotech Inc RayBio Rat Cytokine Antibody Array G series 21
3. 9 6016 6023 Struzynska L Dabrowska Bouta B Koza K Sulkowski G Inflammation Like Glial Response in Lead Exposed Immature Rat Brain Toxicol Sci 2007 95 1 156 162 Watanabe M Guo W Zou S Sugiyo S Dubner R Ren K Neurosci Lett 2005 382 128 133 RayBio Rat Cytokine Antibody Array G series 19 Newman MB Willing AE Manresa JJ Davis Sanberg C Sanberg PR Stroke induced Migration of Human Umbilical Cord Blood Cells Time Course and Cytokines Stem Cells Devel 2005 14 576 586 Moon SK Woo J I Lee H Y Park R et al Toll Like Receptor 2 Dependent NF kappa B Activation Is Involved in Nontypeable Haemophilus influenzae Induced Monocyte Chemotactic Protein 1 Up Regulation in the Spiral Ligament Fibrocytes of the Inner Ear Infect Immun 2007 75 7 3361 3372 Dave B Eason RR Geng Y Su Y Badger TM Simmen RCM Tp53 Associated Growth Arrest and DNA Damage Repair Gene Expression Is Attenuated in Mammary Epithelial Cells of Rats Fed Whey Proteins J Nutr 2006 136 1156 1160 Togel F Hu Z Weiss K Issac J Lange C Westenfelder C Administered mesenchymal stem cells protect agains ischemic acute renal failure through differentiation independent mechanisms Am J Physiol Renal Physiol 2005 289 F3 1 F42 Ahmed N Dreier R Gopferich A Grifka J Grassel S Soluble Signalling Factors Derived from Differentiated Cartilage Tissue Affect Chondrogenic Differentiation of Rat Adult Marrow Stromal Cells Cell Physiol Biochem
4. Biotech s first protein array system known as RayBio Rat Cytokine Antibody Array is particularly useful compared with the Rat cytokine cDNA microarray system Besides the ability to detect protein expression RayBiotech s system is a more accurate reflection of active cytokine levels because it only detects secreted cytokines and no amplification step is needed Furthermore it is much simpler faster environmentally friendlier and more sensitive Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool to study cytokines Cytokines play an important role in innate immunity apoptosis angiogenesis cell growth and differentiation Cytokines are involved in most disease processes including cancer and cardiac diseases The interaction between cytokines and the cellular immune system is a dynamic process The interactions of positive and negative stimuli and positive as well as negative regulatory loops are complex and often involve multiple cytokines Reference List 1 LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT6 B with effects on multiple cytokines Tang X Marciano DL Leeman SE Amar S PNAS 2005 102 14 5132 5137 2 HIV 1 mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced encephalopathy Xu Y Kulkoshy J Pomerantz RJ PNAS 2004 3101 18 7071 7075 3 Synergistic increases in intracellular Ca 2 and the release of M
5. CP 1 RANTES and IL 6 by astrocytes treated with opiates and HIV 1 Tat El Hage N Gurwell JA Singh IN Knapp PE Nath A Hauser KF GLIA 2005 50 2 91 106 RayBio Rat Cytokine Antibody Array G series 3 4 Bone Marrow Stroma Influences Transforming Growth Factor B Production in Breast Cancer Cells to Regulate c myc Activation of the Preprotachykinin I Gene in Breast Cancer Cells Oh HS Moharita A Potian JG Whitehead IP et al Cancer Res 2004 64 6327 6336 5 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 Osario Y Ghiasi H J Virology 2005 79 6 3297 3308 6 Dysregulated Inflammatory Response to Candida albicans in a C5 Deficient Mouse Strain Mullick A Elias M Picard S Gros P Infect Immunity 2004 72 10 5868 5849 7 Leukotriene B4 Strongly Increases Monocyte Chemoattractant Protein 1 in Human Monocytes Huang L Zhao A Wong F Ayala JM Cui J Arterioscler Thromb Vascul Biol 2004 24 1783 1788 8 Giroux M Denis F Human CD1d unrestricted NKT cells release chemokines upon Fas engagement Blood 2005 105 2 703 710 9 Monitoring the response of orthotopic bladder tumors to granulocyte macrophage colony stimulating factor therapy using the prostate specific antigen gene as a reporter Wu Q Esuvaranathan K Mahendran R Clin Cancer Res 2004 10 20 6977 84 10 Neuroglial activatio
6. RayBio Rat Cytokine Antibody Array G Series Patent Pending Technology User Manual Revised February 18 2008 RayBio Rat Cytokine Antibody Array G Series 1 Cat AAR CYT G1 4 RayBio Rat Cytokine Antibody Array G Series 2 Cat AAR CYT G2 4 RayBio Rat Cytokine Antibody Array G Series 1 Cat AAR CYT G1 8 RayBio Rat Cytokine Antibody Array G Series 2 Cat AAR CYT G2 8 RayBio Custom Rat Cytokine Antibody Array G Series 1 Cat AAR CUST G RayBio Rat Cytokine Antibody Array G Series 1 Service Cat AAR SER G Please read the manual carefully before you start your experiment RayBiotech Inc We Provide You With Excellent Protein Array Systems And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Rat Cytokine Antibody Array G Series Protocol TABLE OF CONTENTS I TO GW COM resort crateraerebeeeniavaadseeisesiacaess 2 How It WorkS c cece cece ee ecee eee ee eee ees 5 II Materials Provided cc ccc ece cece cece nes 6 Additional Materials Required 4 6 III Overview and General Considerations 7 A Preparation of Samples 0ceee eee 7 B Handling Glass Chips 0ceee ees 8 C Incubation asc sceanens vestoareeeseasstesetenewenses 8 IV POU CO seccetctpe ces ane tate nee tater dnenanemaneaaenes 9 A Blocking and I
7. archers until now Our RayBio Rat Cytokine Antibody Array is the first commercially available rat cytokine antibody array system By using the RayBiotech system scientists can rapidly and accurately identify the expression profiles of multiple cytokines in several hours inexpensively The RayBiotech kit G series is a glass chip format The kit provides a highly sensitive approach to simultaneously detect multiple cytokine expression levels from cell culture supernatant patient s serum tissue lysate and other sources The arrays are manufactured using non contact arrayer The experimental procedure is simple and can be performed in any laboratory The signals from G series arrays are detected using a laser scanner Besides the products listed in this manual RayBiotech also provides RayBio Mouse Cytokine Antibody Array G series 2000 for simultaneous detection of 144 mouse cytokines in single experiment RayBiotech also provides RayBio Human Cytokine Antibody Array G series 4000 which is the only product available in the market that can detect 274 human cytokines in single experiment We also provide quantitative antibody arrays which can RayBio Rat Cytokine Antibody Array G series 2 quantitatively measure multiple protein expression in antibody array technology Pathway specific array systems allow investigators to focus on the specific problem and are becoming an increasingly powerful tool in cDNA microarray system Ray
8. incubation may be done at 4 C for overnight 13 Wash with Wash Buffer I twice as directed in steps 5 B Fluorescence Detection 1 Decant excess Wash Buffer from wells RayBio Rat Cytokine Antibody Array G series 11 2 Disassemble the slide out of the incubation frame and chamber 3 Place the whole slide in a 30 ml centrifuge tube provided add enough Wash Buffer I about 20 ml to cover the whole slide and gently shake at room temperature for 10 minutes Decant Wash Buffer I Repeat Wash Buffer I once Wash with Wash Buffer II about 20 ml with gentle shake at room temperature for 10 minutes Or wash using slide chamber Rinse the slide with distilled H O 4 Remove water droplets by centrifuge at 1 000 rpm for 3 minutes and then let slide dry completely in air at least 20 minutes protect from light Make sure the slides are absolutely dry before the scanning procedure 5 Image the signals using laser scanner such as Axon GenePix using cy3 or green channel Excitation frequency 532 nm Note we recommend scanning slides right after experiment You also can store the slide at 20 C in dark for several days If you do not have a laser scanner RayBiotech can provide service for you Just simply send your slide to us and we will take care of it RayBio Rat Cytokine Antibody Array G series 12 V Interpretation of Results The following figure shows RayBio Mouse Cytokine Antibody Array G series 1000 probed wi
9. n cytokines e For cell lysates and tissue lysates we recommend using RayBio Cell Lysis Buffer to extract proteins from cell or tissue e g using homogenizer Dilute 2X RayBio Cell Lysis Buffer with HO we recommend adding proteinase inhibitors to Cell Lysis Buffer before use After extraction spin the sample down and save the supernatant for your experiment Determine protein concentration e We recommend using o 50 100 ul of Conditioned media undiluted or o 50 100 ul of 2 fold to 5 fold diluted sera or plasma or o 10 200 ug of total protein for cell lysates and tissue lysates If you experience high background you may further dilute your sample RayBio Rat Cytokine Antibody Array G series 7 Handling glass chips The microarray slides are sensitive do not touch the surface Hold the slides by the edges only Handle all buffers and slides with powder free gloves Avoid breaking glass slide Handle glass chip in clean environment C Incubation Completely cover array area with sample or buffer during incubation and cover the incubation chamber with adhesive film or plastic sheet protector to avoid drying Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rotation Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or 50 ul of sample or reagent is used Avoid cross contamination from overflowing solu
10. n and neuroinflammation in the brain of patients with autism p NA Vargas DL Nascimbene C Krishnan C Zimmerman AW Pardo CA Ann Neurol 2005 57 1 67 81 11 Cytokine profiling of macrophages exposed to Porphyromonas gingivalis its LPS or its FimA Zhou Q Desta T Graves DT Amar S Infect Immunity 2005 73 2 935 43 12 Veto like activity of mesenchymal stem cells functional discrimination between cellular responses to alloantigens and recall antigens Rameshwar P Journal of Immunology 2003 171 7 3426 34 13 The promise of cytokine antibody arrays in drug discovery process Huang RP Yang W Yang Y Flowers L et al Expert Opinion on Drug Discovery 2005 9 601 615 RayBio Rat Cytokine Antibody Array G series 4 Here s how it works Array Ea support axy Sample lt Incubation of Sample f j YYY With arrayed antibody 1 2 hrs S t Cocktail of i y a ae TEPOS Biotin Ab K Sy Incubation with Biotinylated Ab 1 2 hrs Labeled 4 aaaa streptavidin E Incubation with vYY Labeled streptavidin i j Y Detection of signals Data analysis and graph JI I RayBio Rat Cytokine Antibody Array G series II Materials Provided Upon receipt all components of the RayBio Rat Cytokine Antibody Array kit should be stored at 20 C At 20 C the kit will retain complete activity for up to 6 months Once thawed the glass chips Fluo
11. ncubation 0068 9 B IDGLCCHON ir23psitsesatecseuluttsacntniationcerisae s 12 V Interpretation of Results c cece eee 13 VI Troubleshooting Guide cc cee eee es 18 VII Selected References Using RayBiotech Products 19 Cytokine Antibody Arrays are RayBiotech patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Rat Cytokine Antibody Array G series 1 I Introduction All cell functions including cell proliferation cell death and differentiation as well as maintenance of health status and development of disease are controlled by many genes and signaling pathways New techniques such as cDNA microarrays have enabled us to analyze the global gene expression However almost all cell functions are executed by proteins which cannot be studied by DNA and RNA alone Experimental analysis clearly shows a disparity between the relative expression levels of mRNA and their corresponding proteins Therefore it is critical to analyze the protein profile Currently two dimensional polyacrylamide SDS page coupled with mass spectrometry is the mainstream approach to analyzing multiple protein expression levels However the requirement of sophisticated devices and the lack of quantitative measurements greatly limit its broad application Thus no simple cost effective and rapid method of analysis of multiple protein expression levels has been available to rese
12. r 10 min per wash Decant the 1X Wash Buffer I from each well Put the glass chip with frame into the box with 1X Wash Buffer II cover the whole glass slide and frame with Wash Buffer II and wash 2 times at room temperature with gentle shaking for 5 min per wash Remove all of Wash Buffer I in the well Dilute 20X Wash Buffer I with H O RayBio Rat Cytokine Antibody Array G series 10 8 Prepare working solution for biotin conjugated antibodies After brief spin to force antibody aliquot to bottom of tube add 300 ul of Ix blocking buffer to the Biotin Conjugated Antibody tube Mix gently Note the diluted biotin conjugated antibodies can be stored at 4 C for 2 3 days 9 Add 70 ul of diluted biotin conjugated antibodies to each subarray Incubate at room temperature for 2 hours Note incubation may be done at 4 C for overnight 10 Wash as directed in steps 5 and then wash 3 times with 150 ul of 1X Wash Buffer II at room temperature with shaking 2 min per wash Completely remove wash buffer II in each wash step 11 Add 70 ul of 1 500 fold diluted Fluorescent dye conjugated streptavidin after brief spin to force liquid to bottom of tube add 1 5 ml of Blocking Buffer to Fluorescent dye conjugated streptavidin tube to each subarray Cover the incubation chamber with Adhesive film Cover the plate with aluminum foil to avoid exposure to light or incubate in dark room 12 Incubate at room temperature for 1 to 2 hours Note
13. reference 13 in the introduction Positive control Positive control is biotinylated protein It can be used to normalize the streptavidin incubation step If the positive signals from different array membranes are similar positive control is a simple and effective way for normalization Negative control Negative control is BSA Normally it should only give a background reading Data Extraction Tips e Ignore any comet tails e Define the area for signal capture for all spots as 110 120 micron diameter using the same area for every spot e Use median signal value not the total or the mean e Use local background correction also median value e Exclude obvious outlier data in its calculations Using these guidelines along with using PMT brightness and contrast settings that reduce the background as much as possible we get very good results with interassay and intraassay CV lt 20 even with some imperfections in the antibody spots Threshold of significant difference in expression Any gt 1 5 fold increase or lt 0 65 fold decrease in signal intensity for a single analyte between samples provided that both signals are well above background Mean background 2 standard deviations accuracy 95 RayBio Rat Cytokine Antibody Array G series 14 RayBio Rat Antibody Array G Series 1 Detect 19 cytokines in one experiment a b c d e f g h OO RAON RayBio Rat Cytokine Antibody Array G series 2 for simul
14. rescent dye streptavidin Internal Control and 2X Blocking Buffer should be kept at 20 C and all other component should be stored at 4 C Use within three months after reagents have been thawed Please use within six months of purchase e RayBio Rat Cytokine Antibody Microarray slides 1 slide with 4 or 8 subarrays each e Biotin Conjugated Anti Cytokines 1 tube 4 subarrays or 2 tubes 1 500X Fluorescent Dye conjugated Streptavidin Cy3 equivalent 1 tube 2X Blocking Buffer 10 ml 20X Wash Buffer I 30 ml 20X Wash Buffer II 30 ml 2X Cell Lysis Buffer 10 ml RayBio G series antibody array accessory including slide incubation chamber Gasket Protective cover Snap on sides and adhesive film 30 ml tube e Manual Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water e Plastic box RayBio Rat Cytokine Antibody Array G series 6 Layout of Rat G series BU a Antibody Antibody J 9 Array Array E E E BU EE Blak gt _ O Blank gt L mim OU OO EE Bareow Ipil Barcode IIl 4 arrays in one glass chip 8 arrays in one glass chip III Overview and General Considerations A Preparation of Samples e Use serum free conditioned media if possible e If serum containing conditioned media is required use uncultured media as a negative control sample since many types of sera contai
15. t Insufficient wash Increase wash time and use more wash buffer Bubbles formed dunng Avoid bubble formation incubation during incubation Arrays are not completed Completely cover arrays Covered by reagent with solution RayBio Rat Cytokine Antibody Array G series 18 VII Selected References Using RayBiotech Products Human AB Serum and Thrombin Activated Platelet Rich Plasma Are Suitable Alternatives to Fetal Calf Serum for the Expansion of Mesenchymal Stem Cells from Adipose Tissue Kocaoemer A Kern S Kluter H Bieback K Stem Cells 2007 25 1270 1278 ATP Binding by Monarch 1 NLRP12 Is Critical for Its Inhibitory Function Ye Z Lich JD Moore CB Duncan JA Williams KL Ting JP Y Mol Cell Biol 2008 28 1841 1850 Infiltration of CD4 lymphocytes into the brain contributes to neurodegeneration in a mouse model of Parkinson disease Brochard V Combadie re B Prigent A Laouar Y et al J Clin Invest 2009 119 182 192 Transferrin derived synthetic peptide induces highly conserved pro inflammatory responses of macrophages Haddad G Belosevic M Mol Immunol 2009 46 576 586 Robinson K Vona Davis L Riggs D Jackson B McFadden D Peptide YY Attenuates STAT1 and STAT3 Activation Induced by TNF alpha in Acinar Cell Line AR42J J Am Coll Surg 2006 2002 788 796 Stokes L Surprenant A Purinergic P2Y2 Receptors Induce Increased MCP 1 CCL2 Synthesis and Release from Rat Alveolar Peritoneal Macrophages J Immunol 2007 17
16. taneous detection of 34 rat cytokines IL 13 Leptin LIX L Selectin MCP 1 MIP 3alpha PDGF AA Prolactin R Thymus TIMP 1 Chemokine 1 Abbreviations IP 10 Interferon inducible protein 10 LAP latency associated peptide TGF 1 LIF leukocyte inhibitory factor MMP Matrix Metalloproteinase Pos positive control Neg negative control All other are used standard abbreviations RayBio Rat Cytokine Antibody Array G series 15 RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical community RayBio s patent pending technology allows detection of over 180 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services 1 Antibody arrays Cytokine antibody array Human cytokine antibody arrays Mouse cytokine antibody arrays Rat cytokine antibody arrays Pathway or disease focused antibody arrays Inflammation antibody array Angiogensis antibody array Chemokine antibody array Growth factor antibody array MMP antibody array Atherosclerosis antibody array Quantibody arrays for quantitative measurement of cytokine and other protein concentraton Phosphorylation antibody arrays Biotin label based antibody arrays for high density antibody arrays Antibody
17. th different cell culture supernatant The images were captured using laser scanner The biotin conjugated protein produces positive signals which can be used to identify the orientation and to compare the relative expression levels among the different wells The signal intensities obtained from laser scanner can simply be imported into our analysis tool The analysis tool will help you Locate your signal intensities to antibody array map Link the protein to website for more detailed information on the particular protein Protein list sorting Average signal intensities Subtract background Normalize the data from different samples Obtain protein level comparison charts among different samples This analysis tool is very simple and affordable which will not only assist in compiling and organizing your data but also reduces your calculations to a copy and paste step Conditioned medium Negative Control If you do not use our RayBio Analysis Tool you can locate the cytokines by referring to RayBio Rat cytokine Antibody Array G series 1 RayBio Rat Cytokine Antibody Array G series 13 Normalization and comparison For biomarker discovery or for analysis of large number of arrays great attention must be paid to the normalization Our antibody array design includes several controls for normalization and comparison of arrays performing in different membranes and different experiments for more information please read the
18. tion to neighboring wells Several incubation steps such as step 3 blocking step 4 sample incubation step 9 biotin Ab incubation or step 12 Fluorescent dye streptavidin incubation may be done at 4 C for overnight Please make sure to cover the incubation chamber tightly to prevent evaporation RayBio Rat Cytokine Antibody Array G series 8 IV Protocol A Blocking and Incubation 1 Take the glass chip out from the box Let air dry for 2 hours 2 Assemble the glass chip into incubation chamber and incubation frame as shown below Note if you slide has be assembled you can go to step 3 directly Instructions for incubation chamber assembly G Series and Q series arrays Incubation chamber gt gt Gasket normally _ _ gt attached to chamber Protective Cover can ae ad be discarded Seay Snap on sides ee Carefully place slide at bottom of the chamber 7 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 muun While gently holding chamber and slide place side on chamber as shown beginning with N 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 RayBio Rat Cytokine Antibody Array G series 9 3 Add 100 ul 1 X Blocking Buffer into each
19. well and incubate at room temperature for 30 min to block slides Dilute 2X Blocking Buffer with H O Make sure no bubbles are in the well Note only add reagents to wells printed with antibodies 4 Decant Blocking Buffer from each well Add 50 to 100 ul of each sample to array III and array IV Incubate arrays with sample at room temperature for 1 to 2 hours Dilute sample using 1X Blocking Buffer if necessary Note Incubation may be done overnight at 4 C Note We recommend using 50 to 100 ul of undiluted conditioned media or 50 to 100 ul of 2 5 fold diluted serum or plasma or 10 200 ug of protein for cell lysates and tissue lysates Dilute the lysate at least 10 fold with 1X blocking buffer to make a total volume of 50 to 100 ul Make sure there are no bubbles in the wells Note The amount of sample used depends on the abundance of cytokines More concentrated sample can be used if signals are too weak If signals are too strong the sample can be diluted further Decant the samples from each well and wash 3 times with 150 ul of 1X Wash Buffer I at room temperature with gentle shaking 2 min per wash Dilute 20X Wash Buffer I with HO Completely remove wash buffer I in each wash step Note avoid solution flowing into neighboring wells 6 Put the glass chip with frame into a box with 1X Wash Buffer I cover the whole glass slide and frame with Wash Buffer I and wash 2 times at room temperature with gentle shaking fo
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