Human Cytokine Antibody Array G series 6
Contents
1. Arrav 6 Array 7 Array 8 Array 9 Array 10 BI Bi O LI EO aray MO Oo MU ML mO MO INU 3JUu mI EL BINMII MO OEO MU ma ILI O0 DIE DLD Ogo OO 00 00 oo OO OO LOO 00 OO QQ Barcode III ill DIE C DEI DMI 4 arravs in one glass chip Arrav 6 Array 7 Array 8 Array 9 Array 10 BN EN man Ee array i BN oo Ee ao Lu EH N EJ B BE BN EN oo BN Bak FLILIJ OU 00 OOlII OO OO 00 00 OO OO OO 00 00 00 O0 Barcode IHE HH Caa C ITI 8 arrays in one glass chip A Preparation of Samples e Use serum free conditioned media if possible III Overview and General Considerations e If serum containing conditioned media is required use uncultured media as a negative control sample since many types of sera contain cytokines RavBio Human Cytokine Antibody Array G series 4000 7 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 H2O 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 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 lysa2. RavBio Human Cytokine Antibody Array G series 4000 5 II Materials Provided Upon receipt all components of the RavBio Human 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 RavBio Human Cytokine Antibody Microarray slides 5 slides with 4 or 8 subarrays each Human 6 7 8 9 and 10 Biotin Conjugated Anti Cytokines 1 tube 4 subarrays 1 2 tubes each Human 6 7 8 9 and 10 1 500X Fluorescent Dye conjugated Streptavidin Cy3 equivalent 1 tube per 20 subarrays 1 or 2 tubes 2X Blocking Buffer 10 ml 20X Wash Buffer I 30 ml X 2 20X Wash Buffer II 30 ml X 2 Internal control powder 1 tube 2X Cell Lysis Buffer 10 ml RavBio G series antibodv arrav accessorv 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 RavBio Human Cytokine Antibody Array G series 4000 6 Lavout of G series 4000
3. Note Incubation may be done at 4 C for overnight Note We recommend using 50 to 100 ul of 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 1 X 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 IX 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 1 X 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 each 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 RavBio Human Cytokine Antibody Array G series 4000 10 with gentle shaking for 5 min per each wash Remove all of Wash Buffer II in the well Dilute 20X Wash Buffer II with H5O Prepar
4. 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 f 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 RavBio Human Cytokine Antibody Array G series 4000 3 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 J Virology Mar 2005 Vol 79 No 6 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 7 Leukotriene B 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 8 Human CD1d unrestricted NKT 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 9 Monitoring the response of orthotopic bladder tumors to granulocyte macrophage colony
5. TNF alpha TNF beta TPO b Erythropoietin R EPO R Fas Ligand Fas TNFRSF6 LEPTIN OB TRAIL R1 IL 1 R4 ST2 LH P Cadherin TRAIL R2 IF IL 1 sRII LIGHT IL 10 LIMPII SR B2 PDGF R beta NG ieti BTC E Selectin IL 1 sRI LI PDGF R alpha TRAIL R3 TRAIL R4 Trappin 2 Elafin Fer RIIB C Carbonic Anhvdrase IX CA9 Ferritin Cardiotrophin 1 CT 1 FGF 4 Cathepsin S FGF 6 CCL14a HCC 1 IL 10 R alpha Lipocalin 2 NGAL TREM 1 IL 10 R beta L Selectin PDGF BB TROY IL 12 p40 LYVE 1 PECAM 1 Platelet Factor 4 PIGF TSH TSLP u PAR CCL21 6ckine Procalcitonin Calcitonin Ubiquitin 1 CCL28 VIC FLRG IL 13 Rat MCP 2 Prolactin VCAM 1 CD14 FIt 3 Ligand IL 13 Ra2 MCP 3 PSA free VE Cadherin CD23 Fc epsilon RII Follistatin Fractalkine MCP 4 PSA total P selectin VEGF VEGF R2 FSH RAGE VEGF R3 CDA0 Ligand Galectin 7 MICA RANTES VEGF D CEA GCP 2 GCSF IL 12 p70 Marapsin Pancreasin IL 13 MCP 1 IL 15 IL 16 IL 17 L 17F IL 17R Resistin GDF 15 MIC 1 IL 18 BPa RayBio Human Cytokine Antibody Array G series 4000 1g 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
6. 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 analysis tool software ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Peptide Protein arrays EIA geo qr T RayBiotech also provides excellent custom service 1 Antibody arrays 2 Protein arrays 3 Peptide synthesis RayBio Human Cytokine Antibody Array G series 4000 19 Production of recombinant protein and antibodv Peptide arravs Phosphorvlation arravs ELISA EIA ere eh Six 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 Arra
7. dve conjugated streptavidin after brief spinning 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 to 2 hours Note incubation may be done at 4 C for overnight 13 Wash with Wash Buffer I twice as directed in steps 5 B Fluorescence Detection Decant excess Wash Buffer from wells Disassemble the slide out of the incubation frame and chamber 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 H5O 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 scann
8. 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 10 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 11 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 12 Veto like activity 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 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 Human Cytokine Antibody Array G series 4000 4 Here s how it works Arrav f gu a support aa o Sample c Incubation of Sample f YYY With arrayed antibody 1 2 hrs Cocktail of PUDDOMS il o Biotin Ab KAKX Y K KX Incubation with Y Y Biotinylated Ab 1 2 hrs Labeled Besa l streptavidin AK Incubation with 2 hrs ha a d Labeled streptavidin i s gt Detection of signals i Data analvsis and graph
9. 00 2 RavBio Human Cvtokine Antibodv Arrav is particularlv useful compared with the human cvtokine cDNA microarrav svstem Besides the abilitv to detect protein expression RavBiotech s svstem 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 Xiaoren Tang Deborah Levy Marciano Susan E Leeman and Salomon Amar PNAS April 5 2005 vol 102 no 14 5132 5137 2 HIV 1 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
10. Gromova Molecular Cellular Proteomics April 2004 11 3 328 39 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 Antimicrobial Agents Chemotherapy 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 Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells Katherine L Meyer Siegler BMC Cancer 2004 4 34 Airway cytokine expression measured by means of protein array in exhaled breath condensate Correlation with physiologic properties in asthmatic patients Matsunaga K Yanagisawa S Ichikawa T Ueshima K etal J Allergy Clin Immunol 2006 188 84 90 In Vivo Proteomic Analysis of Cytokine Expression in Laser Capture Microdissected Urothelial Cells of Obstructed Ureteropelvic Junction Procured by Laparoscopic Dismembered Pyeloplasty Journal of Endourology 2003 June Volume 17 Number 5 Page 333 336 RavBio Human Cytokine Antibody Array G series 4000 23 Note RavBio is the trademark of RavBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology This product is intended for research only and is not to be used for
11. RavBio Human Cvtokine Antibodv Arrav G Series 4000 Patent Pending Technologv User Manual Revised Februarv 18 2009 Combination of Human Cytokine Antibody Array G Series 6 7 8 9 amp 10 Cat AAH CYT G4000 4 RavBio Human Cytokine Antibody Array G Series 6 Cat AAH CYT G6 4 RavBio 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 RavBio Human Cytokine Antibody Array G Series 9 Cat AAH CYT G9 4 RavBio Human Cytokine Antibody Array G Series 10 Cat AAH CYT G10 4 Combination of Human Cytokine Antibody Array G Series 6 7 8 9 amp 10 Cat AAH CYT G4000 8 RavBio Human Cytokine Antibody Array G Series 6 Cat AAH CYT G6 8 RavBio Human Cytokine Antibody Array G Series 7 Cat AAH CYT G7 8 RavBio Human Cytokine Antibody Array G Series 8 Cat AAH CYT G8 8 RavBio Human Cytokine Antibody Array G Series 9 Cat AAH CYT G9 8 RavBio Human Cytokine Antibody Array G Series 10 Cat AAH CYT G10 8 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 RavBio Human Cytokine Antibody Array G Series 4000 Protocol TABLE OF CONTENTS I Intrioductioti c ozone eoe oi eno roter hend 2 How It M OBES
12. 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 RayBio Human Cytokine Antibody Array G series 4000 24 This product is for research use only 2009 RayBiotech Inc RavBio Human Cytokine Antibody Array G series 4000 25
13. e 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 Antibodv 6 tube Mix gentiv Add 300 ul of IX blocking buffer to the Biotin Conjugated Antibodv 7 tube Mix gentiv Add 300 ul of IX blocking buffer to the Biotin Conjugated Antibody 8 tube Mix gentiv Add 300 ul of IX blocking buffer to the Biotin Conjugated Antibodv 9 tube Mix gentiv Add 300 ul of IX blocking buffer to the Biotin Conjugated Antibodv 10 tube Mix gentiv 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 70 ul of diluted biotin conjugated antibodies 6 to each subarray 6 70 wl of diluted biotin conjugated antibodies 7 to each subarray 7 70 ul of diluted biotin conjugated antibodies 8 to each subarray 8 70 ul of diluted biotin conjugated antibodies 9 to each subarray 9 and 70 ul of diluted biotin conjugated antibodies 10 to each subarray 10 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 RavBio Human Cytokine Antibody Array G series 4000 11 11 Add 70 ul of 1 500 fold diluted Fluorescent
14. er RayBiotech 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 4000 12 V Interpretation of Results The following figure shows RavBio 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 the 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 Wifi Arrav VI Array VII Array VIII Patient Serum 1 Patient Serum 2 Patient Serum 3 Negative Control RavBio Human Cytokine Antibody Array G ser
15. er 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 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 HIV 1 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 Veto like activity 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 Monocyte Chemotactic Protein 1 Mediates Prostate Cancer Induced Bone Resorption Lu Y Cai Z Xiao G Keller ET et al Cancer Res 2007 67 8 3646 3653 Proteomic Characterization of the Interstitial Fluid Perfusing the Breast Tumor Microenvironment A Novel Resource for Biomarker and Therapeutic Target Discovery Julio E Celis Pavel Gromov Teresa RayBio Human Cytokine Antibody Array G series 4000 22 Cabezon Jos M A Moreira Noona Ambartsumian Kerstin Sandelin Fritz Rank and Irina
16. ies 4000 13 If you do not use our RayBio Analysis Tool you can locate the cytokines by referring to RayBio Human cytokine Antibody Array G series 2000 Please keep in mind that G series 2000 consists of three individual arrays human cytokine antibody array 6 human cytokine antibody array 7 and human cytokine antibody array 8 Refer to corresponding 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 please read the reference 13 from 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
17. incubation chamber assembly G Series and Q series arrays Incubation chamber _ Gasket normally attached to chamber Protective Cover can In d be discarded Carefully place slide at bottom of the chamber as shown The slide will adhere somewhat to the bottom Warning the slide is fragile so do not apply more than gentle force to the apparatus While gently holding chamber and slide place side on chamber as shown beginning with bottom flap first Then press the top of the side into grove on chamber and then apply even gentle pressure from one end to the other Repeat this procedure with the other side 3 Add 100 ul 1 X Blocking Buffer into each 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 RavBio Human Cytokine Antibody Array G series 4000 9 Note onlv add reagents to wells printed with antibodies 4 Decant Blocking Buffer from each well Add 50 to 100 ul of each sample to subarray 6 subarray 7 subarray 8 subarray 9 and subarray 10 Incubate arrays with sample at room temperature for 1 to 2 hours Dilute sample using 1X Blocking Buffer if necessary We strongly recommend including Internal Control IC in your assay Add 100 ul 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 ul pipettor
18. instream approach to analyzing multiple protein expression levels 77 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 RavBio 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 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 The RavBio Human Cytokine Antibody Array G series 4000 is the only product available in the market that can detect 274 human cytokines in single experiment 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 RayBio Human Cytokine Antibody Array G series 40
19. m the following list and we will produce the customized array at an affordable price For more information please visit our website www raybiotech com RavBio Human Cytokine Antibody Array G series 4000 17 Human Custom Antibodv Arrav List 285 pr 4 1BB TNFRSF9 Cripto 1 oteins GITR MIP 1 alpha MIP 1 beta SCFR CRP Adiponectin Acrp30 CTACK CCL27 Adipsin Factor D CTLA 4 AFP CXCL16 DAN GITR Ligand GM CSF IL 2 IL 2 R beta Growth Hormom MIP 1 delta MIP 3 alpha MIP 3 beta MMP 1 SDF 1 alpha SDF 1 beta sgp130 ShhN Siglec 5 Decorin Angiogenin DKK 1 Angiopoietin 1 DKK 3 HB EGF HCC 4 CCL16 IL 22 MMP 7 MMP 8 Siglec 9 sTNF RII STNT RI Angiopoietin 2 DKK 4 1L29 IFN lambda 1 MMP 9 TACE DPPIV CD26 HVEM IL 3 TARC DR6 k 1 309 IL 31 MPIF 1 TECK CCL25 TGF alpha Axl E Cadherin ICAM 2 IL 5 MSP a Chain TGF beta 1 B7 1 CD80 EDA A2 ICAM 3 IL 5 R alpha NAP 2 TGF beta 2 AR amphiregulin Dt E GF IFN gamma IL 6 TGF beta 3 EGFR BCMA TNFRSF17 EG VEGF PK1 BDNF ENA 78 beta IG H3 Endoglin Betacellulin BTC Endostatin IGFBP 1 IL 6 sR Thyroglobulin Nidogen 1 Entactin Tie IGFBP 4 NT 3 lera luo NRG bela HRGt bota 1 rei bFGF Eotaxin BLC Eotaxin 2 BMP 4 Eotaxin 3 EpCAM TROP1 IGFBP 5 IL 9 R IGFBP 6 Insulin IP 10 LAP TGF b1 Leptin R NT 4 Oncostatin M Osteopontin Osteoprotegerin TIMP 4
20. o2 und eec IE PER E ba is SOR PE 5 Il Materials Provided ssueeeeuuss 6 Additional Materials Required 6 III Overview and General Considerations 7 A Preparation of Samples 7 B Handling Glass Chips ee 8 C Incubatiofi aeo E bm e a ne ta 8 IVe POCO CO NT HT 9 A Blocking and Incubation 9 B Detection ossis id pp dae f 10 V Interpretation of Results seen 13 VI Troubleshooting Guide L 0 cece eee ee 21 VII Selected References Using RayBiotech Products 22 Cytokine Antibody Arrays are RayBiotech patent pending technology RavBio is the trademark of RayBiotech Inc RayBio Human Cytokine Antibody Array G series 4000 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 T 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 ma
21. tes 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 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 RavBio Human Cytokine Antibody Array G series 4000 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
22. 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 RayBio Human Cytokine Antibody Array G series 4000 14 Using these guidelines along with using PMT brightness and contrast settings that reduce the background as much as possible we get verv good results with interassay and intraassav 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 Array Map Notes o lIL I2reacts with both IL 12p40 and IL 12p70 o IL 12p70 only recognizes IL 12p70 o GRO reacts with CXCLI CXCL2 and CXCL3 GRO alpha beta and gamma respectively o GRO alpha reacts only with CXCL I o VEGF reacts with VEGF 165 and VEGF 121 o TGF beta 1 reacts only with active form of TGF beta 1 o AIL MMP antibody pairs detect both pro and mature forms Abbreviations IP 10 Interferon inducible protein 10 LAP latency associated peptide TGF f1 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 C
23. y G series 4000 20 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 EM Ensure sufficient incubation Short incubation times Tie ancl sample 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 biotinvlated 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 environment Insufficient wash Increase wash time and use more wash buffer Bubbles formed during Avoid bubble formation incubation during incubation Arrays are not completed Completely cover arrays Covered by reagent with solution RavBio Human Cytokine Antibody Array G series 4000 21 VII Selected References Using RavBiotech Products 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 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 Kocaoem
24. ytokine Antibody Array G series 6 60 mana ees vore vr et MIP 1 delta MIP 1 delta RavBio Human Cytokine Antibody Array G series 4000 15 Human Cytokine Antibody Array G series 7 60 p oietin 2 reg n in 2 regulin TNFRSF6 iew Fas GRO ICAM 1 scams rapa IGFBP 6 aan ae IL LRI gna IL 12p40 IL 12p70 ia EE pha ICAM 1 ICAM 3 crap IGFBP 6 Igi sk a RA ST2 IL 1 RI IL 11 IL 12 p40 IL 12 p70 PON tactin alpha beta protegerin tactin alpha lbeta protegerin Human Cytokine Antibody Array G series 8 54 B D E F G H I J K L M N zm B7 1 Cardio DR6 B7 1 DRG IL 10 R d IL 18 BP E EH IL 2 R IL 2R IL H IL 10R IL 13 alpha IL 18 BP IL 18 R wwe IL 2 R IL 2 R beta 2 alpha beta beta gamma E BINE re sos or je co ws ers rn SDF 1 7 DGF AA PDGF AB SR TGF alpha TGF beta Tie 1 Tie 2 TIMP 4 c f g h i j k Furin Galectin 7 GDF 15 ee IL 10 R alpha IL 22 IL 28A IL29 IL 31 Insulin cem Hormon hormone Furin Galectin 7 GDF 15 ee IL 10 R alpha IL 22 IL 28A IL29 IL 31 Insulin Bhi lust Hormon hormone rer mer om ee vows Dec wow wee er es es 9 Teppnz L TREMI rue vomi vearo xeu RavBio Human Cytokine Antibody Array G series 4000 16 RayBio Human Cytokine Antibody Array G series 10 49 Angiostatin ANGPTLA Bate2 M mm me s i We also offer Custom Human Cytokine Antibody Arrays You can select the cytokines of interest fro
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