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Contents
1. Human Angiogenesis Antibody Array G Series 1000 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 diagnosis Biotechniques 2005 38 S9 S15 8 Bartek2. Background Subtraction 16 D Normalization of Array Data 16 E Threshold of Significance 17 VI Antibody Array Map 19 VII Troubleshooting Guide cccssssesssseeseeeeeee 21 VIII Selected References 23 RayBio Cytokine Antibody Arrays are patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Human Angiogenesis Antibody Array G Series 1000 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 hormones or neurotransmitters play important roles in inflammation inn
3. 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 Human Angiogenesis Antibody Array G Series 1000 B Handling Glass Slides e Do not remove glass slide from assembly until Step 16 e Hold the slides by edges only do not touch the surface e Handle all buffers and slides with powder free gloves e Dry glass slide completely before proceeding to Step 3 e Handle and dry glass slide in clean environment e Avoid breaking glass slide when removing 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 Slide 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 S
4. 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 Human Angiogenesis Antibody Array G Series 1000 8 Obtain a clean container eg pipette tip box or slide staining jar and place glass slide assemblies into the container Add enough 1X Wash Buffer to submerge both glass slides with chamber assemblies intact approx 30 50 ml and remove all bubbles in wells Wash 10 min at RT with gentle rocking or shaking 9 Remove glass slide assemblies from container and invert to decant liquid Decant buffer from container and replenish with 1X Wash Buffer Submerge both chamber assemblies and wash 10 min at RT with gentle rocking or shaking 10 Remove glass slide assemblies from container and invert to decant liquid Decant buffer from container Repeat Steps 8 amp 9 with Wash Buffer Il 11 Remove glass slide assemblies from container and invert to decant liquid then carefully aspirate wash buffer from wells touching only the corners with your pipette tip Important Note Be sure to use the correct Anti Cytokine mixture with the correct array slide e g use Human ANG G7 Anti Cytokine mixture for the sl
5. 5 IL 17F MIP 1a SDF 1 CK b 8 1 GDNF IL 17R MIP 1B SDF 1B RayBio Human Angiogenesis Antibody Array G Series 1000 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 antibody 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 Human Angiogenesis Antibody Array G Series 1000 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 i
6. 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 Carefully 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 slide during incubations Be sure to completely remove all bubbles from slide 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 21 RayBio Human Angiogenesis Antibody Array G Series 1000 Randomly scattered high intensity spots Dust or other particulates Dry slides in laminar flow hood and or use clean containers and powder free gloves 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 Streptav
7. G NEG NEG NEG NEG NEG Notes on Array Map e ANG Angiogenin bFGF basic FGF FGF2 PLGF Placental Growth Factor THPO Thrombopoietin e GRO detects GRO a GRO B and GRO y CXCL1 CXCL2 amp CXCL3 e TGF B1 reacts only with active form of TGF B1 e VEGF A detects VEGF 165 aa and VEGF 121 aa 19 RayBio Human Angiogenesis Antibody Array G Series 1000 B RayBio Human Angiogenesis Antibody Array G Series 2 Detects 23 human angiogenic factors in one experiment A B C D E F G H 1 POS1 POS2 POS3 NEG NEG ANGPT1 ANGPT2 ANGSTN 2 POS1 POS2 POS3 NEG NEG ANGPT1 ANGPT2 ANGSTN 3 COL18A1 G CSF GM CSF 1 309 IL 10 IL 1a IL 16 IL 2 4 COL18A1 G CSF GM CSF 1 309 IL 10 IL 1a IL 18 IL 2 5 IL 4 l TAC MCP3 MCP4 MMP1 MMP9 PECAM1 Tie 2 6 IL 4 I TAC MCP3 MCP4 MMP1 MMP9 PECAM1 Tie 2 7 TNF a uPAR VEGF R2 VEGF R3 NEG NEG NEG NEG 8 TNF a uPAR VEGF R2 VEGF R3 NEG NEG NEG NEG Notes on Array Map e ANGPT1 Angiopoietin 1 ANGPT2 Angiopoietin 2 ANGSTN Angiostatin COL18A1 Endostatin Tie 2 TYRO3 e VEGF R2 and VEGF R3 recognize soluble forms of these receptors RayBio Human Angiogenesis Antibody Array G Series 1000 20 VII Troubleshooting guide Problem Cause Recommendation No signal for any spots including Positive Controls Global detection failure Adjust scanner settings or re assemble slide into holder wash slide 2 x 5 min with 150 ul Wash
8. G Series 1000 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 volume 50 100 ul per sub array Cell Cultured Media Neat no dilution needed 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 ul 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
9. IF 1 TGFa Angiopoietin 2 Dtk ICAM 1 IL 31 MSPa TGFB1 Angiostatin E Cadherin ICAM 2 IL 4 NAP 2 TGFB2 ANGPTL4 EDA A2 ICAM 3 IL 5 NCAN 1 TGFB3 Axl EGF IFNy IL 5 Ra NGF R TPO B7 1 EGFR IGF 1 SR IL 6 Nidogen 1 Thyroglobulin BCAM EG VEGF IGFBG 1 IL 6 sR NrCAM Tie 1 BCMA ENA 78 IGFBP 2 IL 7 NRG1 81 Tie 2 BDNF Endoglin IGFBP 3 IL 8 NT 3 TIM 1 B2M Eotaxin IGFBP 4 IL 9 NT 4 TIMP 1 B IG H3 Eotaxin 2 IGFBP 6 Insulin Oncostatin M TIMP 2 bFGF Eotaxin 3 IGF I IP 10 Osteopontin TIMP 4 BLC Ep CAM IGF I SR I TAC OPG TNFa BMP 4 ErbB2 IGF II LAP PAI I TNFB BMP 5 ErbB3 IL 1a Leptin PARC TNFRSF21 BMP 6 EPOR IL 16 Leptin R PDGF Ra TNFRSF6 BMP 7 E Selectin IL 1 R Il LIF PDGF RB TRAIL R2 B NGF Fas IL 1 R4 ST2 LIGHT PDGF AA TRAIL R3 BTC Fas Ligand IL 1 RI LIMPII PDGF AB TRAIL R4 CA125 Fcr RIIB C IL 1 sRI L Selectin PDGF BB Trappin 2 CA15 3 Ferritin IL 10 LH PECAM 1 TREM 1 CA19 9 FGF 4 IL 10 Ra Lymphotactin PIGF TSH CA IX FGF 6 IL 10 RB LYVE 1 PF4 TSLP Cardiotrophin 1 FGF 6 IL 11 Marapsin Procalcitonin Ubiquitin Cathepsin S FGF 7 IL 12 MCP 1 Prolactin uPAR CCL14a FGF 9 IL 12 p40 MCP 2 PSA free VCAM 1 CCL21 Fit 3 Ligand IL 12 p70 MCP 3 PSA total VE Cadherin CCL 28 FLRG IL 13 MCP 4 RAGE VEGF CD14 Follistatin IL 13 Ra 2 M CSF RANK VEGF R2 CD23 Fractalkine IL 13 RI M CSF R RANTES VEGF R3 CD30 FSH IL 15 MDC Resistin VEGF C CD40 Furin IL 16 MICA S 100b VEGF D CD40 Ligand Galectin 7 IL 17 MICB SAA XEDAR CD80 GCP 2 IL 17B MIF SCF CEA G CSF IL 17C MIG SCF R CEACAM 1 GDF 1
10. 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 Human Angiogenesis Antibody Array G Series 1000 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 slides 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 accidentally stored at room temperature RT f
11. RayBio Human Angiogenesis Antibody Array G Series 1000 Patent Pending Technology User Manual Revised June 6 2014 RayBio Human Angiogenesis Antibody Array G Series 1000 Cat AAH ANG G1000 4 RayBio Human Angiogenesis Antibody Array G Series 1000 Cat AAH ANG G1000 8 RayBio Human Cytokine Antibody Array G Series Testing Service Cat AAH SERV G Please read manual carefully before starting experiment Ra 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 slide 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 I
12. ate 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 Human Angiogenesis Antibody Array G Series 1000 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 Sensitivity As little as 4 pg ml of MCP 1 can be detected using t
13. canning and Data Extraction Tips see also page 18 For tips on scanning and data extraction please visit our Website http www raybiotech com Tech Support ScanningTips pdf For a list of recommended scanners please visit our Website http www raybiotech com files T ech Support Laser Scanners for Glass Slide Arrays pdf RayBio Human Angiogenesis Antibody Array G Series 1000 IV Protocol A Preparation 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 1 Blocking Buffer is supplied at 1X concentration No dilution is required 2 Wash Buffers and Il are supplied at 20X concentration a For each glass slide 4 or 8 sub arrays slide dilute 6 ml of 20X concentrate with deionized H20 to a final volume of 120 ml each of Wash Buffer amp Wash Buffer Il b 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 3 Biotin conjugated Anti Cytokines are supplied at high concentration in a small liquid bead typically 2 5 ul a Spin down each tube prior to reconstitution as the concentrated liquid bead may have moved to the top of the tube during handling b Prepare each stock reagent by adding 300 ul 1X Blocking Buffer to Biotin Conjugated Anti Cytokines Mix well c 1X Biotin Conjugated Anti Cytokines may be s
14. cterial challenge impairs their recruitment J Leuk Biol 2012 published online before print doi 10 1189 jlb 0312112 Ye Z Lich JD Moore CB Duncan JA Williams KL Ting JP Y ATP Binding by Monarch 1 NLRP 12 Is Critical for Its Inhibitory Function Mo Cell Biol 2008 28 1841 1850 Dumortier J Streblow DN Moses AV Jacobs JM et al Human Cytomegalovirus Secretome Contains Factors That Induce Angiogenesis and Wound Healing J Virol 2008 82 13 6524 6535 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 2010 9 1085 1099 23 RayBio Human Angiogenesis Antibody Array G Series 1000 Customized RayBio Cytokine Antibody Arrays Select your 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 4 1BB CNTF GITR IL 18 BPa MIP 16 SAA ACE 2 Cripto GITR Ligand IL 18 RB MIP 3a sgp130 Acrp30 CRP GM CSF IL 1ra MIP 3B Shh N Activin A CTACK GRO IL 2 MMP 1 Siglec 5 Adiposin CXCL16 GROa IL 2 RB MMP 10 Siglec 9 Adipsin DAN GH IL 2 Ry MMP 13 ST2 AgRP Decorin HB EGF IL 2 Ra MMP 2 sTNF RI ALCAM Dkk 1 HCC 4 IL 21R MMP 3 sTNF RII a Fetoprotein Dkk 3 hCG intact IL 22 MMP 7 TACE Amphiregulin Dkk 4 HGF IL 28A MMP 8 TARC Angiogenin DPPIV HVEM IL29 MMP 9 TECK Angiopoietin 1 DR6 1 309 IL 3 MP
15. he 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 slide 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 of this easy to use array format see Section VIII RayBio
16. ide designated as ANG G1 not ANG G2 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 from each well 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 chambers with Adhesive film included in kit then cover entire assemblies with aluminum foil to avoid exposure to light or incubate in dark room Incubate at RT for 2 hours with gentle rocking or shaking 13 RayBio Human Angiogenesis Antibody Array G Series 1000 15 Remove aluminum foil and adhesive film Carefully aspirate Streptavidin Fluor reagent from each well 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 16 Remove the glass slides from the frame assemblies Place both slides in 30 ml centrifuge tube provided or slide staining jar Add enough Wash Buffer to cover both slides 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 1 18 Decant buffer and repeat
17. ided 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 obiain quantitative data ie 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 Use local background correction also median value Exclude obvious outlier data in its calculations Scan all slides at same PMT 18 RayBio Human Angiogenesis Antibody Array G Series 1000 VI Antibody Array Maps for Human Angiogenesis Array G 1000 A RayBio Human Angiogenesis Antibody Array G Series 1 Detects 20 human angiogenic factors in one experiment A B C D E F G H 1 POS1 POS2 POS3 NEG NEG ANG EGF ENA 78 2 POS1 POS2 POS3 NEG NEG ANG EGF ENA 78 3 bFGF GRO IFN y IGF1 IL 6 IL 8 Leptin MCP1 4 bFGF GRO IFN y IGF1 IL 6 IL 8 Leptin MCP1 5 PDGF BB PLGF RANTES TGF B1 TIMP1 TIMP2 THPO VEGF A 6 PDGF BB PLGF RANTES TGF 81 TIMP1 TIMP2 THPO VEGF A 7 VEGF D NEG NEG NEG NEG NEG NEG NEG 8 VEGF D NEG NE
18. idin Fluor Re assemble slide 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 concentration 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 22 RayBio Human Angiogenesis Antibody Array G Series 1000 Ill Selected References Citing RayBio Human G Series Arrays 1 Mahadevan D Choi J Cooke L Simons B Riles C et al Gene expression and serum cytokine profiling of low stage CLL identify WNT PCP FIt 3L FIt3 and CKCL9 CXCR5 regulators of cell proliferation survival and migration Human Gene Proteom 2009 453634 doi 10 4061 2009 453634 Fruend A Patil CK Campisi J p38MAPK is a novel DNA damage response independent regulator of senescence associated secretory phenotype EMBO J 2011 30 1536 1548 Laberge R M Zhou L Sarantos MR Rodier F Freund A et al Glucocorticoids suppress selected components of the senescence associated secretory phenotype Aging Cell 2012 DOI 10 1111 1474 9726 2012 00818 x Long C Hosseinkhani MR Wang Y Sriramarao P Walcheck B ADAM17 activation in circulating neutrophils following ba
19. in sealed plastic envelope NOTE In some cases 2 slides x 4 sub arrays slide 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 D Additional Materials Required Small plastic boxes or containers Pipettors pipette tips and other common lab consumables Orbital shaker or oscillating rocker Aluminum foil Gene microarray scanner or similar laser fluorescence scanner see pages 9 amp 15 RayBio Human Angiogenesis Antibody Array G Series 1000 E How It Works co Array support YYYYY m Pi Samples Incubation of Samp y le Y with arrayed antibody supports 1 2 hrs cea AAA K K Incubation with Biotinylated Ab 12 hrs Labeled o ooo 4 streptavidin ie Incubation with 1 hrs labeled Streptavidin t Zoe 7 iia signals t i Data analysis a and graph Ill Helpful Tips and General Considerations A Preparation and Storage of Samples 1 General Considerations Freeze samples as soon as possible after collection Avoid multiple freeze thaw cycles If possible sub aliquot your samples prior to initial storage Spin samples hard 5 10 minutes at 10K to 15K RPM immediately prior to incubation of samples with array Optimal sample concentrations may need to be determined empirically based on the signal intensities of spots and background signals obtained RayBio Human Angiogenesis Antibody Array
20. ll 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 then aspirate remaining Blocking Buffer from each well VOTE 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 slide 5 Add 50 to 100 ul of sample to each sub array Be sure to load each sample on 2 slides Angiogenesis Array G1 and G2 Cover the incubation chambers with Adhesive film included in kit Incubate arrays with sample at RT for 2 hours Dilute sample using 1X Blocking Buffer if necessary RayBio Human Angiogenesis Antibody Array G Series 1000 6 Remove adhesive film and carefully aspirate samples from sub arrays touching only the corners with your pipette tip NOTE Try to keep samples from flowing into neighboring wells 7 Wash 3 x 2 min with 150 ul 1X Wash Buffer at RT Be sure to completely remove liquid each time and use fresh buffer for each wash Decant final wash solution before proceeding to next step Instructions for incubation chamber assembly G Series and Quantibody Arrays Incubation chamber Gasket normally attached to chamber Protective Cover can LL be discarded dm li gt Enp IAEA a Carefully place slide at bottom of the chamber as shown The slide will adhere somewhat to
21. n 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 Human Angiogenesis Antibody Array G Series 1000 26
22. nflammation Antibody Arrays o MMP 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 IMrodU CHOON asses civicsiscsacavardadeetsicunencrarsectatasen viaiiigstiesiattadaduas 1 ll Product INFOrMAtION cece teteescssseetettteecsnsneneteeeesnnennnees 5 A Storage Recommendations 0ce ee 5 B RayBio G Series Glass Slide Layout 5 C Materials Provided 0 cece 6 D Additional Materials Required 000c 6 E How lt WOrksS esisiini 7 Ill Helpful Tips and General Considerations 7 A Preparation and Storage of Samples 8 B Handling Glass Slid s cn ecccceeeeeennnin 9 C Incubations and Washes 9 D Data Extraction Tips 9 IV FOC ON eee e ard E 9 A Preparation and Storage of Reagents 10 B Blocking and Incubations 11 C Fluorescence Detection 14 V Interpretation of Results 15 A Explanation of Control Spots 15 B Typical Results using G Series Arrays 15 C
23. or AAH CTY G2000 8 and processed using this standard protocol gl Array VI Array VII Array VIII Patient Serum 1 Patient Serum 2 Patient Serum 3 Negative Control 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 signals before proceeding to analysis C Background Subtraction Most laser fluorescence scanner software can 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 16 RayBio Human Angiogenesis Antibody Array G Series 1000 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 Sof
24. or up to 24 hours B RayBio G Series Glass Slide Layout BO Array 1 Bu gm BL Blank _ _ O L O L Barcode jjI E Array 2 C E Blank gt C O O O O Barcode j II 4 arrays in one glass chip Array 1 Blank gt Barcode RayBio Human Angiogenesis Antibody Array G Series 1000 O O 0O AA ee O O 0O DA E E Array 2 Blank gt UIUUSS A A OOO AA A A Barcode 8 arrays in one glass chip C Materials Provided AAH ANG AAH ANG Item Description G1000 4 G1000 8 RayBio Human Angiogenesis 1slidewith4 1 slidewith8 AAH ANG G1 Array G1 Glass Slide Sub arrays Sub arrays RayBio Human Angiogenesis 1slidewith4 1 slide with 8 AAH ANG G2 Array G2 Glass Slide Sub arays Sub arrays 0103002 Biotin Conjugated Human iea Dea HANG G1 ANG G1 Anti Cytokines 0103002 Biotin Conjugated Human es Dea HANG G2 ANG G2 Anti Cytokines 1 500X HiLyte Plus 555 ical Streptavidin Fluort Nga ee 0103004 B_ 1X Blocking Buffer 20 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 slide with either 4 or 8 printed sub arrays per slide
25. tored for 2 3 days at 4 C 4 Streptavidin Fluor is supplied at 1500x concentration a Mix the tube containing 1500X Streptavidin Fluor well before use as precipitants may form during storage b Add 100 ul of 1X Blocking Buffer to tube containing 1500X Streptavidin Fluor Mix well c Quantitatively transfer all of Streptavidin Fluor reagent from the original tube to a larger one and dilute with 1X Blocking Buffer to a final volume of 1500 ul ie 1 5 ml d Wrap tube containing Streptavidin Fluor with aluminum foil e This working dilution can be stored for 3 5 days at 4 C 10 RayBio Human Angiogenesis Antibody Array G Series 1000 B Blocking and Incubations NOTE Please carefully read Section II of this manual before proceeding NOTE Prepare all reagents immediately prior to use as described above Section IV A and before proceeding 1 Remove packaged glass slides from freezer Place unopened packages on the benchtop Wait for about 15 min for entire chamber assembly to equilibrate to room temperature RT Then open packages remove the chamber assemblies and place them in laminar flow hood to dry for 1 2 hours NOTE Be sure each glass slide is completely dry before proceeding 2 If necessary assemble glass slides into incubation chamber and frame as shown below and 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 we
26. tware 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 normalize 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 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 17 RayBio Human Angiogenesis Antibody Array G Series 1000 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 prov
27. 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 each array All other variables being equal Positive Control intensities should 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 can normalize results in PCR or Western blots respectively Negative Control NEG spots are printed with a protein containing buffer 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 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 15 RayBio Human Angiogenesis Antibody Array G Series 1000 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
28. 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 or you may scan at a later time You may store the slides at RT indefinitely provided they are protected from strong light RayBio Human Angiogenesis Antibody Array G Series 1000 Note Unlike most Cy3 fluors the HiLyte Plus Fluor 555 very stable at RT and resistant to photobleaching However please protect glass slides from strong light and temperatures above RT 23 Scan the glass slides 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
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