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1. Ligand gamma Du pue em n oe m em om m n Lr Ln CN CA merae p mre perennes e pum mers er soci vac renes ese m m p um pom D E sen F poietin regulin TNFRSF6 TNFRSF6 alpha IGFBP 6 6 wa ra st2 IL 1 RI IL 11 11 12 p40 11 12 p70 IL 6R 1 Osteo Oncostatin lbeta protegerin me won pres e p we A B D E F G H 1 L M N B7 1 Cardio DR6 B7 1 Cardio DR6 ig alpha beta beta gamma ET 11 18 BP IL 18 R IL 2 R c Umm win um men pee wes oma ums os beta beta beta ECC SIEHE EE YESFR2 vecrRa wee nes ne we nes Cadherin RayBio Human Cytokine Antibody Array G series 15 Human Cytokine Antibody Array series 9 51 LYVE 1 MICA MICB MMP 2 MMP 7 E NrCAM LIMPII LYVE 1 MICA MICB MMP 2 MMP 7 8 10 NCAM 1 Nidogen i NrCAM RayBio Human Cytokine Antibody Array series 10 49 b f g h i j k 1 POS1 POS2 POS3 4 1BB ACE 2 Alpha fetoprotein Angiopoietin 1 ANGPTL4 Bate2 M 2 POS1 POS2 E HE EN aw 1BB a 2 Atpna fet2. Human Cytokine Antibody Array G Series Patent Pending Technology User Manual Revised February 18 2009 RayBio Human Cytokine Antibody Array G Series 3 Cat AAH CYT G3 4 RayBio Human Cytokine Antibody Array Series 5 Cat AAH CYT G5 4 RayBio Human Cytokine Antibody Array Series 6 Cat AAH CYT G6 4 RayBio Human Cytokine Antibody Array G Series 7 Cat AAH CYT G7 4 RayBio Human Cytokine Antibody Array G Series 8 Cat AAH CYT G8 4 RayBio Human Cytokine Antibody Array G Series 7 Cat AAH CYT G9 4 RayBio Human Cytokine Antibody Array G Series 8 Cat AAH CYT G10 4 RayBio Human Inflammation Antibody Array 3 Cat AAH INF G3 4 RayBio Human Growth Factor Antibody Array Cat AAH GF G1 4 RayBio Human Cytokine Antibody Array G Series 3 Cat AAH CYT G3 8 RayBio Human Cytokine Antibody Array Series 5 Cat AAH CYT G5 8 RayBio Human Cytokine Antibody Array G Series 6 Cat AAH CYT G6 8 RayBio Human Cytokine Antibody Array G Series 7 Cat AAH CYT G7 8 RayBio Human Cytokine Antibody Array G Series 8 Cat AAH CYT G8 8 RayBio Human Cytokine Antibody Array Series 7 Cat AAH CYT G9 8 RayBio Human Cytokine Antibody Array G Series 8 Cat AAH CYT G10 8 RayBio Human Inflammation Antibody Array 3 AAH INF G3 8 RayBio Human Growth Factor Antibody Array AAH GF G1 8 RayBio Custom Human Cytokine Antibody Array Cat AAH CUS G RayBio Human Cytokine Antibody Array Service C
3. XWash Buffer 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 II in the well Dilute 20X Wash Buffer II with 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 VI 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 corresponding well Incubate at room temperature for 2 hours Note incubation may be done overnight at 4 C 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 spinning add 1 5 ml of Blocking Buffer to Fluorescent dye RayBio Human Cytokine Antibody Array G series 10 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 incubation may be done overnight at 4 C 13 Wash with Wash Buffer I twice as directed in steps 5 B Fluorescence De
4. Selectin IL 1 sRI LIF PDGF R alpha TRAIL R3 CA125 Fas Ligand IL 1 sRII LIGHT Fas TNFRSF6 IL 10 LIMPII SR B2 PDGF R beta TRAIL R4 Trappin 2 Elafin Carbonic Anhydrase IX CA9 Fcr RIIB C IL 10 R alpha Lipocalin 2 NGAL Ferritin IL 10 R beta L Selectin PDGF BB TREM 1 TROY Cardiotrophin 1 CT 1 FGF 4 PERENNE Lymphotactin PECAM 1 TSH Cathepsin S FGF 6 IL 12 p40 LYVE 1 Platelet Factor 4 TSLP CCL14a HCC 1 PIGF u PAR CCL21 6ckine CCL28 VIC 1 IL 12 p70 Marapsin Pancreasin 1 1 FLRG IL 13 MCP 2 CD14 Fit 3 Ligand IL 13 Ra2 MCP 3 CD23 Fc epsilon RII Follistatin MCP 4 Fractalkine Procalcitonin Calcitonin Prolactin PSAfree PSA total P selectin Ubiquitin 1 VCAM 1 VE Cadherin VEGF VEGF R2 CD40 Ligand CEA L L IL L L L L L RAGE RANK RANTES Resistin S 100b VEGF R3 VEGF C VEGF D 11 13 15 16 17 17B 17C 17R GDF 15 MIC 1 RayBio Human Cytokine Antibody Array G series 18 SAA 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 Re
5. Conjugated Anti Cytokines 1 tube per 4 arrays 1 tube for 4 subarrays 2 tubes for 8 subarrays 1 500X Fluorescent Dye conjugated Streptavidin Cy3 equivalent 1 tube 2X Blocking Buffer 10 ml 20X Wash Buffer I 30 ml 20X Wash Buffer 30 ml Internal control powder 1 tube 2X Cell Lysis Buffer 10 ml RayBio series antibody array accessory including slide incubation chamber Gasket Protective cover Snap on sides and adhesive film 30 ml tube Manual Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water Plastic box RayBio Human Cytokine Antibody Array G series 6 Layout of G series BO B EN Ban Bank C OO OO OO Barcode Barcode 4 arrays in one glass chip 8 arrays in one glass chip III Overview and General Considerations A Preparation of Samples Use serum free conditioned media if possible If serum containing conditioned media is required use uncultured media as a negative control sample since many types of sera contain cytokines 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 we recommend adding proteinase inhibitors to Cell Lysis Buffer before use After extraction spin the
6. SV 1 Challenge JOURNAL OF VIROLOGY Mar 2005 Vol 79 No 6 Dysregulated Inflammatory Response to Candida albicans in a C5 Deficient Mouse Strain Alaka Mullick Miria Elias Serge Picard Philippe Gros Infection and Immunity Oct 2004 p 5868 5876 DOI 10 1128 IAI 72 10 5868 5876 2004 Leukotriene B4 Strongly Increases Monocyte Chemoattractant Protein 1 in Human Monocytes Li Huang Annie Zhao Frederick Wong Julia M Ayala Jisong Cui Arteriosclerosis Thrombosis and Vascular Biology 2004 24 1783 1788 Human CD1d unrestricted cells release chemokines upon Fas engagement Martin Giroux and Frangois Denis Yan Xu Joseph Kulkoshy Roger j Pomerantz Blood prepublished online September 2 2004 DOI 10 1182 blood 2004 04 1537 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 Clinical Cancer Research 2004 Oct 15 10 20 6977 84 Neuroglial activation and neuroinflammation in the brain of patients with autism p NA Diana L Vargas Caterina Nascimbene Chitra Krishnan Andrew W Zimmerman Carlos A Pardo Annals of Neurology 2005 Jan 1 DOI 10 1002 ana 20315 Cytokine profiling of macrophages exposed to Porphyromonas gingivalis its LPS or its FimA Qingde Zhou Tesfahun Desta Dana T Graves and Salomon Amar Infection and Immunity IAI 2005 Feb 73 2 935 43 Veto like a
7. at AAH 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 2 RayBiotech Inc RayBio Human Cytokine Antibody Array G Series Protocol TABLE OF CONTENTS I IntoductlObl eios 2 How It WY ODES sodas ebbe 5 Materials 6 Additional Materials Required 6 III Overview and General Considerations 7 A Preparation of a Handling Glass Chips 7 C IncubatiOtic 8 IV 9 A Blocking and Incubation 9 audet Odd mua ende ud 11 V Interpretation of 0 65 17 VI Troubleshooting 21 bitu 22 Cytokine Antibody Arrays are RayBiotech patent pending technology is the trademark of RayBiotech Inc RayBio Human Cytokine Antibody Array series 1 I Introduction All cell functions including cell proliferation cell death and differentiation as well as maintenance of health status and deve
8. cer Induced Bone Resorption Lu Y Cai Z Xiao G Keller ET et al Cancer Res 2007 67 8 3646 3653 Antibody array generated profiles of cytokine release from THP 1 leukemic monocytes exposed to different amphotericin B formulations Turtinen LW Prall DN Bremer LA Nauss RE Hartsel SC Antimicrob Agents Chemother 2004 Feb 48 2 396 403 Reduced T cell and dendritic cell function is related to cyclooxygenase 2 overexpression and protaglandin e 2 secretion in patients with breast cancer Pockaj BA Basu GD Annals of Surgical Oncology 3 327 344 2004 Airway cytokine expression measured by means of protein array in exhaled breath condensate Correlation with physiologic properties in asthmatic patients Matsunaga Yanagisawa S Ichikawa T Ueshima et al J Allergy Clin Immunol 2006 188 84 90 Classification and prediction of clinical Alzheimer s diagnosis based on plasma signaling proteins Ray S Britschgi M Herbert C Takeda Uchimura Y et al Nature Med 2007 13 11 1359 1362 Systemic Endocrine Instigation of Indolent Tumor Growth Requires Osteopontin McAllister SS Gifford AM Greiner AL Kelleher SP Saelzer MP et al Cell 2008 133 994 1005 Chemokine Signaling via the CXCR2 Receptor Reinforces Senescence Acosta JC O Loghlen A Banito A Guijarro MV Augert A et al Cell 2008 133 1006 1018 RayBio Human Cytokine Antibody Array G series 22 Note is the trademark of RayBiotech Inc Cytokin
9. combinant 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 analysis tool software ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Peptide Protein arrays EIA Bes OS Me eer qa RayBiotech also provides excellent custom service 1 Antibody arrays 2 Protein arrays 3 Peptide synthesis 4 Production of recombinant protein and antibody RayBio Human Cytokine Antibody Array G series 19 Peptide arrays Phosphorylation arrays ELISA EJA o ee en 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 Human Cytokine Antibody Array G series 20 VI Troubleshooting guide Problem Weak signal Uneven signal Cause Rec
10. ctivity of mesenchymal stem cells functional discrimination between cellular responses to alloantigens and recall antigens Rameshwar P Journal of Immunology 2003 Oct 1 171 7 3426 34 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 Human Cytokine Antibody Array series 4 Here s how it works Array aN support o Sample c Incubation of Sample YYY With arrayed antibody 1 2 hrs Cocktail of ilo Biotin Ab Incubation with YYY Biotinylated Ab 1 2 hrs Labeled streptavidin XX Incubation with YY 7 Labeled streptavidin ee Detection of signals Data analysis and graph RayBio Human Cytokine Antibody Array G series 5 II Materials Provided Upon receipt all components of RayBio Mouse 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 Fluorescent 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 RayBio Mouse Cytokine Antibody Microarray slides 1 slide with 4 or 8 subarrays each Biotin
11. e 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 15 a trademark of AnaSpec Inc RayBio Human Cytokine Antibody Array G series 23 RayBio Human Cytokine Antibody Array G series This product is for research use only 2009 RayBiotech Inc RayBio Human Cytokine Antibody Array G series 25
12. e relative expression levels among the different wells The internal control IC can also be used to normalize the signal intensities among array membranes in different experiments 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 Array VI Array VII Array VIII Patient Serum 1 Patient Serum 2 Patient Serum 3 Negative Control RayBio Human Cytokine Antibody Array G series 12 If you do not use our RayBio Analysis Tool you can locate the cytokines by referring to corresponding RayBio Human cytokine Antibody Array G series map 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
13. er if necessary We strongly recommend including Internal Control IC in your assay Add 100 of Blocking Buffer to IC tube mix well and transfer 1 ul of IC to each well 50 to 100 ul of sample Note when you transfer IC use 0 1 ul to 2 pipettor Note Incubation may be done overnight at 4 C Note We recommend using 50 to 100 of undiluted conditioned media or 50 to 100 of 2 5 fold diluted serum or plasma or 10 200 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 RayBio Human Cytokine Antibody Array G series 9 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 5 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 H2O 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 for 10 min per wash 7 Decant the 1X Wash Buffer I from each well Put the glass chip with frame into the box with
14. erimental 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 Human Cytokine Antibody Array series 1000 for detection of 120 human cytokines in single experiment RayBio Human Cytokine Antibody Array G series 2000 for detection of 174 human cytokines in single experiment and RayBio Human Cytokine Antibody Array series 4000 for detection of 274 human cytokines in single experiment RayBio Human Cytokine Antibody Array G series 2 Pathway specific array systems allow investigators to focus on the specific problem and are becoming an increasingly powerful tool in cDNA microarray system RayBiotech s first protein array system known as Human Cytokine Antibody Array is particularly useful compared with the human 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 lt Cytokines are involved
15. 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 Xiaoren Tang Deborah Levy Marciano Susan E Leeman and Salomon Amar PNAS April 5 2005 vol 102 no 14 5132 5137 2 HIV I mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced encephalopathy Yan Xu Joseph Kulkoshy Roger j Pomerantz PNAS 2004 May 4 2004 Vol 101 No 18 3 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 GLIA 2005 Apr 15 50 2 91 106 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 Hyun S Oh Anabella Moharita Pranela Rameshwar CANCER RESEARCH 64 6327 6336 September 1 2004 RayBio Human Cytokine Antibody Array G series 3 10 11 12 13 Recombinant Herpes Simplex Virus HSV 1 Codelivering Interleukin 12p35 as a Molecular Adjuvant Enhances the Protective Immune Response against Ocular H
16. lopment 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 7 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 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 7 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 researchers until now Our RayBio Human Cytokine Antibody Array is the first commercially available protein 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 slide 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 exp
17. nalyte between samples provided that both signals are well above background Mean background 2 standard deviations accuracy 95 Array Map Notes O IL 12 reacts with both IL 12p40 and IL 12p70 IL 12p70 only recognizes IL 12p70 GRO reacts with CXCLI CXCL2 and CXCL3 GRO alpha beta and gamma respectively GRO alpha reacts only with CXCL1 VEGF reacts with VEGF 165 and VEGF 121 TGF beta 1 reacts only with active form of TGF beta 1 All MMP antibody pairs detect both pro and mature forms Abbreviations IP 10 Interferon inducible protein 10 LAP latency associated peptide 1 LH leutinizing hormone LIF leukocyte inhibitory factor MMP Matrix Metalloproteinase PF plasma factor 4 Pos positive control Neg negative control All other are used standard abbreviations RayBio Human Cytokine Antibody Array G series 3 42 a b d e f g h 1 j k J oso ss eos ew oe ENT OO i CS E Grow res Grow res TARC 1 TARC TGF p1 RayBio Human Cytokine Antibody Array G series 14 RayBio Human Cytokine Antibody Array series 6 60 A B e D E J K L M N Cro ore t mc mme o m m ross
18. ocol A Blocking and Incubation Take the glass chip out from the box Let air dry for 2 hours 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 Gasket normally attached to chamber Protective Cover can E ad be discarded es Snap on sides gt RayBio Human Cytokine Antibody Array G series 8 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 3 Add 100 ul X Blocking Buffer into each well and incubate at room temperature for 30 min to block slides Dilute 2X Blocking Buffer with 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 6 and array 7 Incubate arrays with sample at room temperature for 1 to 2 hours Dilute sample using 1X Blocking Buff
19. ommendation Inadequate detection Check laser power and parameters Inadequate reagent volumes Check pipetters and or improper dilution ensure correct preparation Ensure sufficient incubation Short incubation times Time and change ample incubation step to overnight Too low protein concentration Don t make 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 parameters Insufficient wash Bubbles formed dunng incubation Arrays are not completed Covered by reagent RayBio Human Cytokine Antibody Array G series 21 Work in clean environment Increase wash time and use more wash buffer Avoid bubble formation during incubation Completely cover arrays with solution 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 Monocyte Chemotactic Protein 1 Mediates Prostate Can
20. oprotein Angiopoietin 1 Angeria Bate2 M 3 BCMA beta IG H3 15 3 19 9 Cathepsin S CCL14a CCL21 Cp23 CD40 Ligand Anhydrase IX Carbonic 4 5 beta IG H3 15 3 19 9 hydr Cathepsin S CCL14a CCL21 CD23 CD40 Ligand 5 CEACAM 1 Cripto 1 Decorin DKK 1 DKK 3 DKK 4 DPPIV E Cadherin EG VEGF 6 CEACAM 1 Cripto 1 Decorin DKK 1 DKK 3 DKK 4 DPPIV E Cadherin A2 EG VEGF ide riis in FECE NEC HET Y y un d AM me su mee owe oes Procalcitonin PSA free S 100b Shh N Thyroglobulin Ubiquitin 1 Blank IC1 2 10 Procalcitonin PSA free S 100b Shh N Thyroglobulin Ubiquitin 1 Blank RayBio Human Inflammation Antibody Array series 3 40 A B D E F G H J K L M posi ross nea NEG EOTAXN EOTAXN2 GCSF GMCSF cawi iw 159 posi Pos2 Poss EOTAXN EOTAXN2 GCSF GMCSF IFNy 159 RayBio Human Cytokine Antibody Array G series 16 RayBio Human Growth Factor Antibody Array 1 Detect 41 Growth Factors in one experiment 1 bFGF EGFR FGF 4 FGF 6 2 bFGF EGFR FGF 4 FGF 6 4 We also offer Custom Human Cytokine Antibody Arrays You can select the cytokines of interest from the following list and we will produce the customized array at an affordable price For more information please visit our website www raybiotech com RayBio Human Cytokine An
21. please read the 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 Internal control RayBio antibody arrays also include spiking in protein serving as internal control IC The spiking in proteins do not have cross reactivity with protein in the array It can be used to normalize the entire process 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 e Scan all slides at same PMT 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 RayBio Human Cytokine Antibody Array G series 13 on ou amp Threshold of significant difference expression Any gt 1 5 fold increase or lt 0 65 fold decrease in signal intensity for a single a
22. sample down and save the supernatant for your experiment Determine protein concentration 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 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 RayBio Human Cytokine Antibody Array G series 7 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 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 solution 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 IV Prot
23. tection Decant excess Wash Buffer from wells Disassemble the slide out of the incubation frame and chamber Place the whole slide in 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 about 20 ml with gentle shake at room temperature for 10 minutes Or wash using slide chamber Rinse the slide with distilled H O 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 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 we can provide service for you Just simply send your slide to us and we will take care of it RayBio Human Cytokine Antibody Array G series 11 V Interpretation of Results The following figure shows RayBio Human Cytokine Antibody Array G series 2000 probed with 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 th
24. tibody Array G series 17 Human Custom Antibody Array List 285 proteins 4 1BB TNFRSF9 CNTF IL 18 alpha Cripto 1 GITR MIP 1 alpha MIP 1 beta SCFR GITR Ligand RP Adiponectin Acrp30 CTACK CCL27 MIP 1 delta SDF 1 alpha GM CSF IL MIP 3 alpha SDF 1 beta Adipsin Factor D CTLA 4 lro IL 2 R alpha MIP 3 beta sgp130 AFP CXCL16 DAN Growth Hormom IL 2 R beta MMP 1 Shh Siglec 5 Decorin HB EGF Angiogenin DKK 1 HCC 4 CCL16 Angiopoietin 1 DKK 3 Angiopoietin 2 DKK 4 HGF DPPIV CD26 MMP 7 Siglec 9 sTNF RII IL 28A IFN lambda MMP 8 sTNT RI 1L29 IFN lambda 1 MMP 9 TACE TARC HVEM DR6 1 309 amphiregulin Dtk IL MPIF 1 TECK CCL25 TGF alpha E Cadherin ICAM 2 IL MSP a Chain TGF beta 1 B7 1 CD80 EDA A2 EGF IFN gamma IL 5 3 ICAM 3 IL 5 R alpha NAP 2 TGF beta 2 6 TGF beta 3 EGFR IGFBP 1 IL 6 sR BCMA TNFRSF17 EG VEGF PK1 IL 7 Nidogen 1 Entactin Thyroglobulin Tie 1 IL 8 NrCAM Tie 2 IL 9 beta Endoglin IGFBP 4 NRG1 beta 1 HRG1 beta 1 TIM 1 Betacellulin BTC Endostatin NT 3 IGFBP 5 IL 9 IGFBP 6 Insulin IP 10 bFGF Eotaxin BLC Eotaxin 2 BMP 4 Eotaxin 3 BMP 5 EpCAM TROP1 Leptin R NT 4 Oncostatin M Osteopontin TIMP 4 TNF alpha LAP TGF b1 Osteoprotegerin TNF beta TPO LEPTIN OB TRAIL R1 b NG Erythropoietin R EPO R IL 1 R4 ST2 LH P Cadherin TRAIL R2 BTC E
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