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1. 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 RT for 30 min to block slides NOTE Only add reagents or samples to wells printed with antibodies see diagram on page 6 4 Decant Blocking Buffer then aspirate remaining liquid from each well NOTE To aspirate liquid samples or reagents from wells gently place the pipette tip only in the corners of the well Do not scrape the pipette tip across the surface of the chip 5 Add 50 to 100 ul of each sample to each sub array Cover the incubation chamber with Adhesive film included in kit Incubate arrays with sample at RT for 2 hours Dilute sample using 1X Blocking Buffer if necessary 6 Remove adhesive film and carefully aspirate samples from sub arrays touching only the corners with your pipette tip NOTE Try to prevent solution from flowing into neighboring wells RayBio Human Obesity Array G1 11 7 Wash 3 x 2 min with 150 ul 1X Wash Buffer at RT Be sure to completely remove sample and Wash Buffer each time and use fresh buffer for each wash Decant final wash solution before proceeding to next step 8 Obtain a clean container eg pipette tip box or slide staining jar and place glass chip assembly into the container Add enough 1X Wash Buffer to submerge the entire
2. MCP 1 MCP 2 MCP 3 MCP 4 M CSF M CSF R MDC MICA MICB MIF MIG MIP 1a MIP 18 21 MIP 16 MIP 3a MIP 3B MMP 1 MMP 10 MMpP 13 MMP 2 MMP 3 MMP 7 MMP 8 MMP 9 MPIF 1 MSPa NAP 2 NCAM 1 NGFR Nidogen 1 NrCAM NRG1 B1 NT 3 NT 4 Oncostatin M Osteopontin OPG PAI I PARC PDGF Ra PDGF RB PDGF AA PDGF AB PDGF BB PECAM 1 PIGF PF4 Procalcitonin Prolactin PSA free PSA total RAGE RANK RANTES Resistin S 100b SAA SCF SCF R SDF 1 SDF 1B SAA sgp130 Shh N Siglec 5 Siglec 9 ST2 sTNF RI sTNF RII TACE TARC TECK TGFa TGFB1 TGFB2 TGFB3 TPO Thyroglobulin Tie 1 Tie 2 TIM 1 TIMP 1 TIMP 2 TIMP 4 TNFa TNFB TNFRSF21 TNFRSF6 TRAIL R2 TRAIL R3 TRAIL R4 Trappin 2 TREM 1 TSH TSLP Ubiquitin uPAR VCAM 1 VE Cadherin VEGF VEGF R2 VEGF R3 VEGF C VEGF D XEDAR Testing Services RayBiotech offers full GLP Certified testing services using any of our Array ELISA or EIA products including customized products Send us your samples we send you 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 Peptoid Synthesis amp Library Screening 1O or LS ee S m Technology Transfer Program Have you developed technologies or reagents of interest to the scientific and research community
3. RayBiotech can help you commercialize your technologies reagents and your dreams RayBio Human Obesity Array G1 22 RayBio Cytokine Antibody Arrays are patent pending technology developed by RayBiotech This product is intended for research only and is not to be used for clinical diagnosis Our produces may not be resold modified for resale or used to manufacture commercial products without written approval by RayBiotech Inc Under no circumstances shall RayBiotech be liable for any damages arising out of the use of the materials Products are guaranteed for 6 months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to buyer for any claim relating to products shall be limited to replacement or refund of the purchase price RayBio is a registered trademark of RayBiotech Inc HiLyte Plus is a trademark of Anaspec Inc InnoScan is a registered trademark of Innopsys Inc This product is for research use only 2012 RayBiotech Inc RayBio Human Obesity Array G1 23
4. 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 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 Cytokine responses elicited in endothelial cells after treatment with a specific toxin Jaya Pandey BioCompare Product Review May 13 2004 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 Cabez n Jos M A Moreira Noona Ambartsumian Kerstin Sandelin Fritz Rank and Irina Gromova Molecular Cellular Proteomics April 2004 11 3 328 39 Increased Expression and Secretion of Interleukin 6 in Patients with Barrett s Esophagus Katerina Dvorakova Harinde
5. 2009 182 6460 6469 Pukstadad BS Ryana L Floa TH Stenvika J et al Non healing is associated with persistent stimulation of the innate immune response in chronic venous leg ulcers J Dermatol Sci 2009 59 2 115 122 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 Streblow DN Dumortier J Moses AV Orloff SL Nelson JA Mechanisms of Cytomegalovirus Accelerated Vascular Disease Induction of Paracrine Factors That Promote Angiogenesis and Wound Healing Shenk TE Stinski MF eds Current Topics in Microbiology and Immunology Human Cytomegalovirus Berlin Heidelberg Germany Springer 2008 325 397 415 Nolting T Lindecke A Koutsilie E Maschke M et al Measurement of soluble inflammatory mediators in cerebrospinal fluid of human immunodeficiency virus positive patients at distinct stages of infection by solid phase protein array J Neruovirol 2009 15 5 6 390 400 Pannebaker C Chandler HL Nichols JJ Tear proteomics in keratoconus Mol Vision 2010 16 1949 1957 RayBio Human Obesity Array G1 20 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 ACE 2 Acrp30 Activin A Ad
6. Duncan JA Williams KL Ting JP Y ATP Binding by Monarch 1 NLRP12 is critical for its inhibitory function Mol Cell Biol 2008 28 1841 1850 4 Sommer G Kralisch S Stangl V Vietzke A et al Secretory products from human adipocytes stimulate proinflammatory cytokine secretion from human endothelial cells J Cell Biochem 2009 106 4 729 737 5 Bouazza B Kratassiouk G Gjata B Perie S et al Analysis of growth factor expression in affected and unaffected muscles of oculo pharyngeal muscular dystrophy OPMD patients A pilot study Neuromusc Disorders 2009 19 3 199 206 6 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 655 7 Keren Z Braun Moscovici Y Markovits D Rozin A Nahir M et al Depletion of B lymphocytes in rheumatoid arthritis patients modifies IL 8 anti IL 8 autoantibody network Clin Immunol 2009 doi 10 1016 j clim 2009 07 001 8 Rovin BH Song H Hebert LA Nadasdy T et al Plasma urine and renal expression of adiponectin in human systemic lupus erythematosus Kidney Int 2005 68 1825 1833 9 Duncan JA Gao X Huang MT H O Connor BP Thomas CE et al Neisseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling RayBio Human Obesity Array G1 19 10 11 12 13 14 Activities of the NLRP3 and ASC Containing Inflammasome J Immunol
7. Map Detects 62 cytokines in one experiment A B c D E F G H I J K L M N 1 POS1 POS2 POS3 NEG 4 1BB ace 2 CRP Adipsin AgRP ANGPT ANGPT ANGPTL icip ENA 30 1 2 4 78 2 POS1 POS2 POS3 NEG 4 1BB ace 2 CRP Adipsin AgRP ANGET CANGPT KANGPTE ekp ENA 30 1 2 4 78 FGF IFN IGFBP IGFB IL 1 IL 1 3 Fas 2 GH HCC 4 cin x p GFBP 3 IGF I IGF I sR ia IL 1 beta IL 1 sRI Ra sr2 FGF IFN IGFBP IGFB IL 1 IL 1 4 Fas 2 GH HCC 4 aes i p GFBP 3 IGF I IGF I sR T IL 1 beta IL 1 sRI oe ae IL 6 Insuli Leptin Lympho 5 IL 6 IL 8 IL 10 IL 11 IL 12 IP 10 Leptin LIF McP 1 MCP 3 sR n R tactin elus E i8 ma0 m1 m12 ip10 Leptin 1EPt ur LymPho mcp 1 mcp 3 sR n R tactin MIP 1 MSP PDGF PDGF PDGF a 7 MCSF MIF Peta dipha OPG OSM PAI I PARC yi aE aa RANTES Resistin SAA Mip 1 MSP PDGF PDGF PDGF a 8 MCSF MIF anne OPG OSM PAI I PARC sd AB ae RANTES Resistin SAA 9 sor a SINF STNE sree TGF TIMP TIMP TNE VEGF XEDAR NEG NEG NEG NEG RI RII Beta 1 2 alpha 10 spr 4 SINF STNE Eck TOFS e TME IMP TNE VEGF XEDAR NEG NEG NEG NEG RI RII Beta 1 2 alpha Note IL 12 only recognizes IL 12 p70 Abbreviations GH Growth Hormone IP 10 Interferon inducible protein 10 LAP leukocyte inhibitory factor MMP Matrix Metallopro
8. RayBio G Series Human Obesity Antibody Array 1 Patent Pending Technology User Manual Revised July 1 2014 4 Sample Kit Cat AAH ADI G1 4 8 Sample Kit Cat AAH ADI G1 8 Human Obesity Array G1 Service Cat AAH ADI G1 SERV Please read the manual carefully before you start your experiment RayBiotech Inc We Provide You With Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Human Obesity Antibody Array G1 Protocol TABLE OF CONTENTS Introduction 2 How It VUNG scutes do hence nada tasatiaia teins case 5 Il Product Information oo smsnnsnmerestninenseneeee 6 A Materials Provided 6 C Additional Materials Required 6 E RayBio G Series Glass Chip Layout 6 III Overview and General Considerations 7 A Preparation and Storage of Samples 7 B Handling Glass Chips ssn 8 C Incubations and Washes 8 D Data Extraction Tips 8 IV PRO UNCON yt oh es ee 9 A Preparation and Storage of Reagents 9 B Blocking and Incubations 10 C Fluorescence Detection n 13 V Interpretation of ResUS n 14 A Explanation of Control Spots 14 B Typical Results using G Series Arrays 14 C Background Subtraction 15 D Normalization of Array Data 15 E Threshold of Significance 16 Vi Antibody Array Map 17 VIL Troubleshooting Guide 18 VIII Selec
9. aluminum foil e This working dilution can be stored for 3 5 days at 4 C RayBio Human Obesity Array G1 9 B Blocking and Incubations NOTE Please carefully read Section III of this manual before proceeding NOTE Prepare all reagents immediately prior to use as described above Section IV A and before proceeding 1 Remove the package containing the glass chip assembly from the freezer Place unopened package on the benchtop and allow the glass chip assembly to equilibrate to room temperature RT approx 15 min Open package remove the glass chip assembly and place in laminar flow hood to dry for 1 2 hours NOTE Be sure glass chip is completely dry before proceeding 2 If necessary assemble the glass chip into incubation chamber and frame as shown below Note if you slide is already assembled you can proceed directly to Step 3 Instructions for incubation chamber assembly G Series and Q series arrays Incubation chamber _ Gasket normally _ _ _ gt attached to chamber Protective Cover can ae be discarded SSeS Snap on sides a RayBio Human Obesity Array G1 10 Carefully place slide at bottom of the chamber 7 as shown The slide will adhere somewhat to the bottom Warning the slide is fragile so do not apply more than gentle force to the apparatus side on chamber as shown beginning with bottom flap first While gently holding chamber and slide place
10. ay 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 eg Array VI Array VI Array VIH Patient Serum 1 Patient Serum 2 Patient Serum 3 Negative Control RayBio Human Obesity Array G1 14 In this example sera from several patients were incubated with Human Cytokine Arrays 6 7 amp 8 sold together as Human Cytokine Array G Series 2000 AAH CYT G2000 4 or AAH CTY G2000 8 and processed using this standard protocol The 6 strong signals of the Positive Control spots in the upper left corner are useful for proper orientation of the array image If scanned using optimal scan settings 3 distinct Positive Control signal intensities will be seen POS1 gt POS2 gt POS3 If all of these signals are of similar intensity try increasing or decreasing laser power and or signal gain settings Once you have obtained fluorescence intensity data you should subtract the background and normalize to the Positive Control signals before proceeding to analysis C Background Subtraction Most laser fluorescence scanner software have an option to automaticall
11. diately prior to use and keep working dilutions of all reagents on ice at all times 1 4 Blocking Buffer supplied as 1X No dilution is required Wash Buffers and Il are supplied at 20X concentration a For each glass chip 4 or 8 sub arrays chip dilute 6 ml of 20X concentrate with deionized H20 to a final volume of 120 ml each of Wash Buffer amp Wash Buffer II 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 Biotin conjugated Anti Cytokines are supplied at high concentration ina small liquid bead typically 2 5 pl a Spin down the tube prior to reconstitution as the concentrated liquid bead may have moved to the top of the tube during handling b Prepare stock reagent by adding 300 ul 1X Blocking Buffer to Biotin Conjugated Anti Cytokines Mix well c 1X Biotin Conjugated Anti Cytokines may be stored for 2 3 days at 4 C 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
12. ected from strong light NOTE Unlike most Cy3 fluors the HiLyte Plus Fluor 555 used in this kit is very stable at RT and resistant to photobleaching on completed glass chips However please protect glass chips from strong light and temperatures above RT 23 Scan the glass chip with a laser scanner such as Innopsys InnoScan using cy3 or green channel excitation frequency 532 nm For tips on scanning visit our Website http www raybiotech com Tech Support ScanningTips pdf NOTE If you do not have a laser scanner for a nominal fee you can send your slide to us for scanning and data extraction using Innopsys InnoScan and we will return the results to you Using using alternate protocols RayBio G Series RayBio Human Obesity Array G1 13 arrays are also compatible with Li Cor s Odyssey and other microarray scanners V Interpretation of Results A Explanation of Controls Spots Positive Controls POS1 POS2 POS3 are equal amounts of biotinylated IgGs printed directly onto the array All other variables being equal the Positive Control intensities will be the same for each sub array This allows for normalization based upon the relative fluorescence signal responses to a known control much as housekeeping genes or proteins are used to normalize results in PCR or Western blots respectively Negative Control NEG spots are a protein containing buffer used to dilute antibodies printed on the arr
13. ensitive Simultaneous detection of multiple adipokines undoubtedly provides a powerful tool to study obesity The area of obesity research is getting hotter ever over the past years One of the key driving factor is that adipose tissue is found no longer to be an inert energy storage organ but is emerging as an active participant in regulating physiological and pathologic processes RayBio Human Obesity Array G1 2 Many soluble factors have been identified from the adipose tissue and are so called as adipocytokines or adipokines But adipokines are also expressed in a number of other tissues and organs Because all of these factors can act in an autocrine paracrine and endocrine manner in the organisms adipokines are thought to serve as mediators linking obesity inflammation immunity and other obesity related diseases 17 Without doubt simultaneous detection of multiple adipokines provides a powerful tool to study adipokines References 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 2 and t
14. glass chip with frame intact approx 30 50 ml and remove all bubbles in wells Wash 10 min at RT with gentle rocking or shaking 9 Remove assembled glass chip from container and invert it to decant liquid Decant buffer from container and replenish with 1X Wash Buffer Submerge the entire glass chip assembly and wash 10 min at RT with gentle rocking or shaking 10 Remove assembled glass chip from container and invert it to decant liquid Decant buffer from container and repeat Steps 8 amp 9 with Wash Buffer II 11 Remove assembled glass chip from container and invert it to decant liquid then carefully aspirate wash buffer from wells touching only the corners with your pipette tip 12 Add 70 ul of 1X Biotin conjugated Anti Cytokines to each sub array Cover incubation chamber with Adhesive film included in kit Incubate at RT for 2 hours with gentle rocking or shaking 13 Carefully aspirate all of the Biotin conjugated Anti Cytokine reagent Wash as described in Step 7 above first with Wash Buffer then with Wash Buffer II making sure to completely remove buffer between washes and after final wash 14 Add 70 ul of 1X Streptavidin Fluor to each sub array Cover the incubation chamber with Adhesive film included in kit then cover entire assembly with aluminum foil to avoid exposure to light or incubate in dark room Incubate at RT for 2 hours with gentle rocking or shaking 15 Remove aluminum foil and adhesive f
15. he 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 Gene in Breast Cancer Cells Hyun S Oh Anabella Moharita Pranela Rameshwar CANCER RESEARCH 64 6327 6336 September 1 2004 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 JOURNAL OF 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 Francois Denis Yan Xu Joseph Kulkoshy Roger j Pomerantz Blood prepublished online September 2 2004 DOI 10 1182 blood 2004 04 1537 RayBio Human Obesity Array G1 3 10 11 12 13 14 15 16 17 Monitoring the response of orthotopic bladder tumors to
16. ilm Carefully aspirate the Streptavidin Fluor reagent Wash as described in Step 7 above first with Wash Buffer then with Wash Buffer Il making sure to completely remove buffer between washes and after final wash RayBio Human Obesity Array G1 12 16 Remove the glass chip from the frame assembly Place the whole chip in 30 ml centrifuge tube provided or slide staining jar Add enough Wash Buffer to cover the whole slide about 20 ml and gently rock or shake at RT for 10 min 17 Decant buffer and repeat wash as described in Step 16 1 x 10 min with Wash Buffer 18 Decant buffer and repeat wash as described in Step 16 but this time using Wash Buffer II for only 2 3 minutes 19 Decant buffer remove the glass chip from the tube then gently rinse the slide with de ionized H20 using a plastic wash bottle 20 Remove water droplets by applying suction gently with a pipette tip NOTE Be careful not to touch the array portions of the slide with your pipette tip only touch the sides of the slide C Obtaining Fluorescent Signal Intensities 21 Allow glass chip to dry in a laminar flow hood for 20 minutes or until slide is completely dry Place chip under an aluminum foil tent to protect it from light Make sure the slides are absolutely dry before scanning or storage 22 You may proceed immediately to scanning Step 23 or you may scan at a later time You may store the slides at RT indefinitely provided they are prot
17. iposin Adipsin AgRP ALCAM a Fetoprotein Amphiregulin Angiogenin Angiopoietin 1 Angiopoietin 2 Angiostatin ANGPTL4 Axl B7 1 BCAM BCMA BDNF B2M B IG H3 bFGF BLC BMP 4 BMP 5 BMP 6 BMP 7 B NGF BTC CA125 CA15 3 CA19 9 CA IX Cardiotrophin 1 Cathepsin S CCL14a CCL21 CCL 28 CD14 CD23 CD30 CD40 CD40 Ligand CD80 CEA CEACAM 1 CK b 8 1 CNTF Cripto CRP CTACK CXCL16 DAN Decorin Dkk 1 Dkk 3 Dkk 4 DPPIV DR6 Dtk E Cadherin EDA A2 EGF EGFR EG VEGF ENA 78 Endoglin Eotaxin Eotaxin 2 Eotaxin 3 Ep CAM ErbB2 ErbB3 EPOR E Selectin Fas Fas Ligand Fer RIIB C Ferritin FGF 4 FGF 6 FGF 6 FGF 7 FGF 9 Fit 3 Ligand FLRG Follistatin Fractalkine FSH Furin Galectin 7 GCP 2 G CSF GDF 15 GDNF RayBio Human Obesity Array G1 GITR GITR Ligand GM CSF GRO GROa GH HB EGF HCC 4 hCG intact HGF HVEM 1 309 ICAM 1 ICAM 2 ICAM 3 IFNy IGF 1 SR IGFBG 1 IGFBP 2 IGFBP 3 IGFBP 4 IGFBP 6 IGF I IGF I SR IGF II IL la IL 1B IL 1 R II IL 1 R4 ST2 IL 1 RI IL 1 sRI IL 10 IL 10 Ra IL 10 RB IL 11 IL 12 IL 12 p40 IL 12 p70 IL 13 IL 13 Ra 2 IL 13 RI IL 15 IL 16 IL 17 IL 17B IL 17C IL 17F IL 17R IL 18 BPa IL 18 RB IL 1ra IL 2 IL 2 RB IL 2 Ry IL 2 Ra IL 21R IL 22 IL 28A IL29 IL 3 IL 31 IL 4 IL 5 IL 5 Ra IL 6 IL 6 sR IL 7 IL 8 IL 9 Insulin IP 10 I TAC LAP Leptin Leptin R LIF LIGHT LIMPII L Selectin LH Lymphotactin LYVE 1 Marapsin
18. ly 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 Streptavidin Fluor Re assemble chip into holder wash 2 x 5 min with 150 ul Wash Buffer Il 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 with sample O N at 4 C Increase concentration of and or length of incubation with Biotin conjugated Anti Cytokine add l large volume wash following Biotin Ab incubation Review proper storage conditions for kit components RayBio Human Obesity Array G1 18 Ill Selected References Citing RayBio Human G Series Arrays 1 Mamlouk O Balagurumoorthy P Wang K Adelstein SJ Kassis Al Bystander effect in tumor cells produced by iodine 125 labeled human lymphocytes Intl J Radiation Biol 2012 DOI 10 3109 09553002 2012 702297 2 Kocaoemer A Kern S Kluter H Bieback K 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 Stem Cells 2007 25 1270 1278 3 Yez Lich JD Moore CB
19. ml Slide incubation chamber 8 G Series Array Accessories gasket protective cover snap Room Temperature on sides and adhesive film Additional Materials Required Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water Plastic box Layout of G Series Slides E L Antibody Array Blank gt OOE oodd OO 4 arrays in one glass chip RayBio Human Obesity Array G1 Antibody Array Blank gt JIS O El JIG Barcode 8 arrays in one glass chip Ill Overview 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 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 Final sample volume 50 100 ul per sub array e Cell Cultured Media Neat no dilution needed e Se
20. on E Threshold of significant difference in expression After subtracting background signals and normalization to Positive controls comparison of signal intensities for antigen specific antibody spots between and among array images can be used to determine relative differences in expression levels of each analyte ie protein detected between samples or groups Any 21 5 fold increase or lt 0 65 fold decrease in signal intensity for a single analyte between samples or groups may be considered a measurable and significant difference in expression provided that both sets of signals are well above background Mean background 2 standard deviations accuracy 95 NOTE In the absence of an external standard curve for each analyte there is no means of assessing absolute or relative concentrations of different analytes in the same sample using immunoassays If you wish to obtain quantitative data 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 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 RayBio Human Obesity Array G1 16 VI RayBio Human Obesity Array G1
21. r Garewal Clinical Cancer Research 2004 Mar 15 10 6 2020 8 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 The promise of cytokine antibody arrays in drug discovery process R P Huang W Yang D Yang L Flowers I R Horowitz X Cao and R Huang Expert opinion on drug discovery 2005 9 601 615 RayBio Human Obesity Array G1 4 Here s how it works YYY a Antibody array chip SEn Y Y Y 1 2 hours m P ee pa of sample ESN YYY ae K Incubation with biotinylated Ab Incubation with 1 hour al Streptavidin conjugated Hylite Plus _ gt Data Analysis and graph RayBio Human Obesity Array G1 5 Il Materials Provided Store kit at lt 20 C immediately upon arrival Kit must used within the 6 month expiration date STORAGE TEMPERATURE TEM COMPONENT AAH ADI 1 4 AAH ADI 1 8 AFTER THAWING 1 a Obesity Array G1 Glass habeas Sabanas ON 2 Blocking Buffer 10ml 20ml Human Obesity Array G1 2 8 C Biotinylated Antibody Cocktail on nDe for up to 3 days after dilution 1 500X Fluorescent Dye 4 conjugated Streptavidin Cy3 1 tube equivalent re 5 20X Wash Buffer Concentrate 30ml 6 20X Wash Buffer II Concentrate 30ml 7 2X Cell Lysis Buffer Concentrate 10
22. rum amp Plasma 5 fold to 10 fold dilution e Most other Body Fluids Neat or 2 fold to 5 fold dilution e Cell and Tissue Lysates Minimum 5 fold to 10 fold to equal concentrations of total protein in each lysate sample 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 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 To start we recommend using 10 100 ug of total protein in 100 ul of 1X Blocking Buffer final volume per sub array 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 RayBio Human Obesity Array G1 7 e Other Liquid Sample Types Most often Neat or 2 fold to 5 fold However optimal dilutions should be determined empirically For tips on sample preparation please visit our Website http www raybiotech com Tech Support SampleTips pdf B Handling Glass Chips e Do not remove glass chip 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 glove
23. s e Dry glass chip completely before proceeding to Step 3 e Handle and dry glass chip in clean environment e Avoid breaking glass chip when removing the chamber assembly C Incubations and Washes e 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 e Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle s e 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 e Avoid cross contamination of samples to neighboring wells e To remove Wash Buffers and other reagents from chamber wells you may invert the Glass Chip Assembly to decant and aspirate the remaining liquid e In Wash Steps 6 12 and 15 you may gently flush wells several times using a wash bottle filled with Wash Buffer I D Scanning and Data Extraction Tips For tips on scanning and data extraction please visit our Website http www raybiotech com Tech Support ScanningTips pdf For a list of recommended scanners please visit our Website http www raybiotech com Tech Support Laser Scanners for Glass Slide Arrays pdf RayBio Human Obesity Array G1 8 IV Protocol A Preparation and Storage of Reagents NOTE During this protocol prepare reagents imme
24. sively 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 experimental procedure is simple and can be performed in any laboratory The signals from G series arrays are detected using a laser scanner Besides the products listed in this manual RayBiotech also provides RayBio Human Cytokine Antibody Array G 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 G series 4000 for detection of 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 Obesity Antibody Array is particularly useful compared with the human adipokine cDNA microarray system Besides the ability to detect protein expression RayBiotech s system is a more accurate reflection of active adipokine levels because it only detects secreted adipokines and no amplification step is needed Furthermore it is much simpler faster environmentally friendlier and more s
25. ted References 19 Cytokine Antibody Arrays are RayBiotech patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Human Obesity Array G1 1 l Introduction All cell functions including cell proliferation cell death and differentiation as well as maintenance of health status and development of disease are controlled by many genes and signaling pathways New techniques such as cDNA microarrays have enabled us to analyze the global gene expression However almost all cell functions are executed by proteins which cannot be studied by DNA and RNA alone Experimental analysis clearly shows a disparity between the relative expression levels of mRNA and their corresponding proteins Therefore it is critical to analyze the protein profile Currently two dimensional polyacrylamide SDS page coupled with mass spectrometry is the mainstream approach to analyzing multiple protein expression levels However the requirement of sophisticated devices and the lack of quantitative measurements greatly limit its broad application Thus no simple cost effective and rapid method of analysis of multiple protein expression levels has been available to 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 inexpen
26. teinase SAA Serum Amyloid A Pos positive control Neg negative control latency associated peptide TGF b1 LIF All other are used standard abbreviations RayBio Human Obesity Array G1 17 VII Troubleshooting guide Problem Cause Recommendation No signal for any spots including Positive Controls Global detection failure Adjust scanner settings or re assemble chip into holder wash slide 2 x 5 min with 150 ul Wash Buffer II and repeat Steps 12 21 Similar signal intensities for POS1 2 3 Improper laser power and or PMT setting Repeat scan using higher and or lower laser power or PMT settings High background signals Incomplete washes 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 chip during incubations Be sure to completely remove all bubbles from chip surface Evaporation during incubation steps Cover chamber assembly during washes and incubations Pooling precipitation of sample or reagent Incomplete washes Cover chamber assembly and use a rocker or shaker during washes and incubations carefully follow wash protocols Sample is too concentrated Repeat experiment using more dilute sample Random
27. y measure the local background around each spot As with spot signal intensities we recommend using MEDIAN background signals If your resulting fluorescence signal intensity reports do not include these values eg a column labeled as MED532 B532 you may need to subtract the background manually or change the default settings on your scanner s data report menu D Normalization of Array Data To normalize signal intensity data one sub array is defined as reference to which the other arrays are normalized This choice can be arbitrary For example in our Analysis Tool Software the array represented by data entered in the left most column each worksheet is the default reference array You can calculate the normalized values as follows X Ny X y P1 P y Where P1 mean signal intensity of POS spots on reference array P y mean signal intensity of POS spots on Array y X y mean signal intensity for spot X on Array y RayBio Human Obesity Array G1 15 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 informati

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