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1. 1 Mamlouk O Balagurumoorthy P Wang K Adelstein SJ Kassis Al Bystander effect in tumor cells produced by iodine 125 labeled human lymphocytes nt J Radiation Biol 2012 DOI 10 3109 09553002 2012 702297 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 Ye Z Lich JD Moore CB Duncan JA Williams KL Ting JP Y ATP Binding by Monarch 1 NLRP12 is critical for its inhibitory function Mo Cell Biol 2008 28 1841 1850 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 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 Weuromusc Disorders 2009 19 3 199 206 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 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 C in mmunol 2009 doi 10 1016 j clim 2
2. 1 VCAM 1 VEGF 7 NEG NEG NEG NEG NEG NEG NEG NEG 8 NEG NEG NEG NEG NEG NEG NEG NEG Abbreviations POS Positive Control NEG Negative Control KIM 1 Kidney Injury Molecule 1 ALB Albumin B2M B2 Microglobulin OPN Osteopontin L FABP Liver Fatty Acid Binding Protein HGF Hepatocyte Growth Factor MIF Macrophage migration Inhibitory Factor NGAL Neutrophil Gelatinase Associated Lipocalin Lipocalin 2 All others use standard abbreviations RayBio Human Acute Kidney Injury Antibody Array G Series 1 19 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 Il 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 inc
3. Inflammation Antibody Arrays MMP Antibody Arrays o Obesity Antibody Arrays Quantibody Multiplex ELISA Arrays RayBio L Series Biotin Label based Antibody Arrays RayBio E Series Competition based Antibody Arrays RayBio Phosphorylation Antibody Arrays o Receptor Tyrosine Kinases o EGFR and ErbB family site specific phosphorylation Over 700 different ELISA kits EIA Competitive ELISA kits Cell based Phosphorylation Assay Over 10 000 different antibodies Recombinant proteins Peptide Recombinant antibodies O O O O O O TABLE OF CONTENTS l introduch Oeccneaenisen nikaa 1 Il Product INFOrMAtION cccccseeeessccsssstttesecesssentetseeennneetee 4 A Storage Recommendations cc cece 4 B Materials Provide cesses 5 C Additional Materials Required 5 D How lt Works sacs aas ccecace tis acsedccacateceacreadtalcseaatecticadetashs 6 E RayBio G Series Glass Chip Layout 6 Ill Helpful Tips and General Consideratione 7 A Preparation and Storage of Samplee 7 B Handling Glass Chips 8 C Incubations and WaSN6ES c cee 8 D Data Extraction Tips 9 IV FF MOG Ol dase oe cetectnca eee Cae en ee eter 9 A Preparation and Storage of Reagents 9 B Blocking and Incubations 10 C Fluorescence Detection sscssiiisressi 14 V Interpretation of Results 15 A Explanation of Control Spots 15 B Typical Result
4. J Mori K Barasch J Devarajan P Neutrophil gelatinase associated Lipocalin NGAL as a biomarker for acute renal injury after cardiac surgery Lancet 2005 365 1231 1238 RayBio Human Acute Kidney Injury Antibody Array G Series 1 9 Han WK Bailly V Abichandani R Thadhani R Bonventre JV Kidney Injury Molecule 1 KIM 1 A novel biomarker for human renal proximal tubule injury Kidney Inti 62 237 244 2002 10 Herget Rosenthal S Marggraf G Husing J Goring F Pietruck F Janssen O Philipp T Kribben A Early detection of acute renal failure by serum cystatin C Kidney Inti 66 1115 1122 2004 11 Yamamoto T Noiri E Ono Y Doi K Negishi K Kamijo A Kimura K Fujita T Kinukawa T Taniguchi H Nakamura K Goto M Shinozaki N Ohshima S Sugaya T Renal L type fatty acid binding protein in acute ischemic injury Am Soc Nephrol 18 2894 2902 2007 12 Munshi R Johnson A Siew ED Ikizler TA Ware LB Wurfel MM Himmelfarb J Zager RA MCP 1 gene activation marks acute kidney injury Am Soc Nephrol 2011 Jan 22 1 165 75 13 Yu Y Jin H Holder D Ozer JS Villarreal S Shughrue P Shi S Figueroa DJ Clouse H Su M Muniappa N Troth SP Bailey W Seng J Aslamkhan AG Thudium D Sistare FD Gerhold DL Urinary biomarkers trefoil factor 3 and albumin enable early detection of kidney tubular injury Nat Biotechnol 2010 May 28 5 470 7 Il Product Information A Storage Recommendations For best results we rec
5. 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 Il making sure to completely remove buffer between washes and after final wash 14 Add 70 ul of 1X Streptavidin Fluor to each sub array Cover the incubation 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 film 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 13 RayBio Human Acute Kidney Injury Antibody Array G Series 1 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 describ
6. 009 07 001 22 RayBio Human Acute Kidney Injury Antibody Array G Series 1 8 Rovin BH Song H Hebert LA Nadasdy T et al Plasma urine and renal expression of adiponectin in human systemic lupus erythematosus Kidney nt 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 Activities of the NLRP3 and ASC Containing Inflammasome J mmunol 2009 182 6460 6469 10 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 Sa 2009 59 2 115 122 11 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 Ce Proteom 2010 9 1085 1099 12 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 13 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 s
7. 1X Blocking Buffer final volume per sub array o Optimal amounts of total lysate protein may range from 50 1000 ug per sub array Based upon background and spots intensities you may increase or decrease the amount of protein used in subsequent experiments e Other Liquid Sample Types Most often Neat or 2 fold to 5 fold However optimal dilutions should be determined empirically 3 Sample Preparation For tips on sample preparation please visit our Website http www raybiotech com Tech Support SampleTips pdf B Handling Glass Chips Do not remove glass chip from assembly until Step 16 Hold the slides by edges only do not touch the surface Handle all buffers and slides with powder free gloves Dry glass chip completely before proceeding to Step 3 Handle and dry glass chip in clean environment 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 Il 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 RayBio Hum
8. RayBio Human Acute Kidney Injury Antibody Array 1 G Series Patent Pending Technology User Manual Revised July 2 2012 RayBio Human Acute Kidney Injury Antibody Array G Series Cat AAH AKI G1 4 RayBio Human Acute Kidney Injury Antibody Array G Series Cat AAH AKI G1 8 RayBio Human Cytokine Antibody Array G Series Testing Service Cat AAH SERV G Please read manual carefully before starting experiment RayBiotech Inc the protein array pioneer company FN ii We provide you with excellent Protein Array systems and services Tel Toll Free 1 888 494 8555 or 1 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com Email info raybiotech com RayBiotech Inc the Protein Array Pioneer Company strives to research and develop new products to meet demands of the biomedical community RayBiotech s patent pending technology allows detection of up to 507 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable reproducible and cost effective Our product offerings include wo o N e i a a Protein antigen Arrays RayBio Cytokine Antibody Arrays C Series Membrane chemiluminescence detection G Series Glass chip fluorescence detection Pathway and disease focused antibody arrays o Angiogenesis Antibody Arrays Apoptosis Antibody Arrays Atherosclerosis Antibody Arrays Chemokine Antibody Arrays Growth Factor Antibody Arrays
9. SR IGF II IL 1a IL 1B IL 1 R Il 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 MCP 1 MCP 2 MCP 3 MCP 4 M CSF M CSF R MDC MICA MICB MIF MIG MIP 1a MIP 1B RayBio Human Acute Kidney Injury Antibody Array G Series 1 MIP 15 MIP 3a MIP 3B MMP 1 MMP 10 MMP 13 MMP 2 MMP 3 MMP 7 MMP 8 MMP 9 MPIF 1 MSPa NAP 2 NCAM 1 NGF R 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 PECAN 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 VCAN 1 VE Cadherin VEGF VEGF R2 VEGF R3 VEGF C VEGF D XEDAR 24 Testing Services RayBiotech offers full testing services using any of our Array ELISA or EIA prod
10. 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 4 Streptavidin Fluor is supplied at 1500x concentration a Mix the tube containing 1500X Streptavidin Fluor well before use as precipitants may form during storage b Add 100 ul of 1X Blocking Buffer to tube containing 1500X Streptavidin Fluor Mix well c Quantitatively transfer all of Streptavidin Fluor reagent from the original tube to a larger one and dilute with 1X Blocking Buffer to a final volume of 1500 ul ie 1 5 ml d Wrap tube containing Streptavidin Fluor with aluminum foil e This working dilution can be stored for 3 5 days at 4 C B Blocking and Incubations NOTE Please carefully read Section II of this manual before proceeding NOTE Prepare all reagents immediately prior to use as described above Section IV A and before proceeding 1 Remove 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 proce
11. able 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 lie concentrations of the various analytes in your samples try using our Quantibody Multiplex ELISA arrays instead Data Extraction Tips e Ignore any comet tails Define the area for signal capture for all spots as 110 120 micron diameter using the same area for every spot Use median signal value not the total or the mean Use local background correction also median value Exclude obvious outlier data in its calculations Scan all slides at same PMT 18 RayBio Human Acute Kidney Injury Antibody Array G Series 1 VI RayBio Human Acute Kidney Injury Antibody Array G Series Map Detects 20 cytokines in one experiment A B C D E F G H 1 POS1 POS2 POS3 NEG KIM 1 ALB OPN TFF3 2 POS1 POS2 POS3 NEG KIM 1 ALB OPN TFF3 3 B2M Clusterin CXCL16 GPNMB L FABP MCP 1 sTNFRI Calbindin 1 4 B2M Clusterin CXCL16 GPNMB L FABP MCP 1 sTNFRI Calbindin 1 5 IP 10 CystatinC HGF MIF NGAL TIMP 1 VCAM 1 VEGF 6 IP 10 CystatinC HGF MIF NGAL TIMP
12. an Acute Kidney Injury Antibody Array G Series 1 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 Scanning Tips pdf For a list of recommended scanners please visit our Website http www raybiotech com resources asp See also page 18 of this manual IV Protocol A Preparation and Storage of Reagents NOTE During this protocol prepare reagents immediately prior to use and keep working dilutions of all reagents on ice at all times 1 Blocking Buffer is supplied at 1X concentration No dilution is required 2 Wash Buffers and Il are supplied at 20X concentration a For each glass 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 3 Biotin conjugated Anti Cytokines are supplied at high concentration in a small liquid bead typically 2 5 ul RayBio Human Acute Kidney Injury Antibody Array G Series 1 a
13. ata extraction using Innopsys InnoScan and we will return the results to you Using using alternate protocols RayBio G Series arrays are also compatible with Li Cor s Odyssey and other microarray scanners V Interpretation of Results A Explanation of Controls Spots Positive Controls POS1 POS2 POS3 are equal amounts of biotinylated IgGs printed directly onto 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 array 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 15 RayBio Human Acute Kidney Injury Antibody Array G Series 1 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 i eg Array VI Array VII Array VIII Patient Serum 2 a oe Patient Serum 3 In this example sera from several patients were
14. e 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 Acute Kidney Injury Antibody Array G Series 1 26
15. ed in Step 16 1 x 10 min with Wash Buffer Decant buffer remove the glass chip from the tube then gently rinse the slide with de ionized H20 using a plastic wash bottle 18 Remove excess liquid from 30 ml centrifuge tube provided in kit and place glass chip into the tube Centrifuge at 1 000 rpm for 3 minutes to remove water droplets C Obtaining Fluorescent Signal Intensities 19 Remove slide from tube and allow glass chip to dry in a laminar flow hood for at least 20 minutes Place chip under an aluminum foil tent to protect it from light Make sure the slides are absolutely dry before scanning or storage 20 You may proceed immediately to scanning Step 21 or you may scan at a later time You may store the slides at RT indefinitely provided they are protected from strong light Note Unlike most Cy3 fluors the HiLyte Plus Fluor 532 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 21 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 Scanning Tips pdf 14 RayBio Human Acute Kidney Injury Antibody Array G Series 1 NOTE If you do not have a laser scanner for a nominal fee you can send your slide to us for scanning and d
16. eding 10 RayBio Human Acute Kidney Injury Antibody Array G Series 1 2 f necessary assemble the glass chip into incubation chamber and frame as shown on page 12 Note if you slide is already assembled you can proceed directly to Step 3 3 Add 100 ul 1 X Blocking Buffer into each 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 5 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 Instructions for incubation chamber assembly G Series and Quantibody Arrays Incubation chamber Gasket normally _ gt attached to chamber Protective Cover can ae ais be discarded erty Snap on sides a 11 RayBio Human Acute Kidney Injury Antibody Array G Series 1 6 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 gentl
17. 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 Patient Serum 1 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 RayBio Human Acute Kidney Injury Antibody Array G Series 1 C Background Subtraction Most laser fluorescence scanner software have an option to automatically measure the local background around each spot As with spot signal intensities we recommend using MEDIAN background signals If your resulting fluorescence signal intensity reports do not include these values eg a column labeled as MED532 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 arb
18. itrary 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 X Ny normalized signal intensity for spot X on Array y The RayBio Analysis Tool software is available for use with data obtained using RayBio G Series Arrays You can copy and paste your signal intensity data with and without background into the Analysis Tool and it will automatically normalize signal intensities to the Positive Controls To order the Analysis Tool please contact us at 1 770 729 2992 or info raybiotech com for more information 17 RayBio Human Acute Kidney Injury Antibody Array G Series 1 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 measur
19. monitoring and kidney injury early detection 1 Tang X Marciano DL Leeman SE Amar S LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT6 B with effects on multiple cytokines PNAS 2005 102 14 5132 5137 2 Xu Y Kulkosky J Acheampong E Nunnari G Sullivan J Pomerantz RJ HIV 1 mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced encephalopathy PNAS 2004 101 18 7070 7075 3 El Hage N Gurwell JA Singh IN Knapp PE Nath A Hauser KF 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 Oh HS Moharita A Potian JG Whitehead IP et al 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 Cancer Res 2004 64 6327 6336 5 Bonventre JV Weinberg JM Recent advances in the pathophysiology of ischemic acute renal failure J Am Soc Nephrol 2003 14 2199 210 6 Lameire N Hoste E Reflections on the definition classification and diagnostic evaluation of acute renal failure Editorial Curr Opin Crit Care 2004 10 468 75 7 American Society of Nephrology Renal Research Report J Am Soc Nephrol 16 1886 1903 2005 8 Mishra J Dent C Tarabishi R Mitsnefes MM Ma Q Kelly C Ruff SM Zahedi K Shao M Bean
20. of samples with array e Optimal sample concentrations may need to be determined empirically based on the signal intensities of spots and background signals obtained e Most samples will not need to be concentrated If concentration is required we recommend using a spin column concentrator with a chilled centrifuge Recommended Sample Volumes and Dilution Factors NOTE All sample dilutions should be made using 1X Blocking Buffer For all sample types final sample volume 50 700 ul per sub array Cell Cultured Media Neat no dilution needed Serum amp Plasma 5 fold to 10 fold dilution Most other Body Fluids Neat or 2 fold to 5 fold dilution Cell and Tissue Lysates Minimum 5 fold to 10 fold to equal concentrations of total protein in each lysate sample 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 Total Lysate Protein Concentration prior to sample dilution 5 ug l 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 50 ul to 100 ul final volume RayBio Human Acute Kidney Injury Antibody Array G Series 1 o To start we recommend using 100 200 yg of total protein in 100 ul of
21. ommend storing the entire kit at 20 C or 80 C upon arrival and using the kit within 6 months of receipt RayBiotech warranties this product for 6 months if stored in this manner Once thawed store glass chips and 1X Blocking Buffer at 20 C or 80 C and all other component at 4 C After thawing the entire kit should be used within 3 months RayBio Antibody Array kits are robust and will retain full activity even if accidentally stored at room temperature RT for up to 24 hours RayBio Human Acute Kidney Injury Antibody Array G Series 1 B Materials Provided 7 AAH AKI AAH AKI Item Description G1 4 G1 8 RayBio Human Acute Kidney 1 chip with 4 1 chip with 8 AAH AKI G1 Injury Microarray Glass Chip Sub arrays Sub arrays 0103002 Biotin Conjugated Anti es 9 en HAKI1 Cytokines 1 500X HiLyte Plus 532 UM epee Streptavidin Fluort Nee Nee 0103004 B 1X Blocking Buffer 10 ml 20 ml 0103004 Wt 20X Wash Buffer 30 ml 30 ml 0103004 Wt 20X Wash Buffer II 30 ml 30 ml 0103004 L 2X Cell Lysis Buffer optional 10 ml 10 ml Other Kit Components Manual Adhesive Plastic Strips 30 ml Centrifuge Tube Kit contains 1 pre assembled glass chip with either 4 or 8 printed sub arrays per chip in sealed plastic envelope NOTE In some cases 2 chips x 4 sub arrays chip may be substituted in kits containing 8 sub arrays t This fluor is patent pending technology from Anas
22. onic kidney disease at the time of patient presentation to rapidly manage associated illness However serum creatinine a standard marker of kidney function does not distinguish acute kidney injury from prerenal azotemia or chronic kidney disease In addition the initial measurement of serum Creatinine cannot reflect the extent of injury because its accumulation always lags behind the insult Concurrently the potential for improving risk stratification informing clinical decision making and guiding pharmaceutical development recently led the American Society of Nephrology to designate the development of novel AKI biomarkers a top research priority The response over a few years resulted in the identification of nearly 20 potential markers Some of the more promising of these include urine or plasma Neutrophil Gelatinase associated io ee NGAL Kidney Injury Molecule 1 KIM 1 Cystatin C Liver Fatty acid Binding Protein _ L FABP Monocyte Chemoattractant Protein 1 MCP 1 and Trefoil Factor 3 TFF3 RayBio Human Acute Kidney Injury Antibody Array G Series 1 Traditionally urine proteins or cytokines are detected by using ELISA However RayBio Human Acute Kidney Injury Antibody Array G Series can detect 20 protein biomarkers simultaneously with small amount of sample It is a great tool in the acute kidney injury research areas including drug toxicity monitoring kidney transplantation rejection reaction
23. pec Inc Wash Buffers are sold as sets C Additional Materials Required e Small plastic boxes or containers e Pipettors pipette tips and other common lab consumables e Orbital shaker or oscillating rocker e Aluminum foil e Gene microarray scanner or similar laser fluorescence scanner see pages 9 amp 15 RayBio Human Acute Kidney Injury Antibody Array G Series 1 D How It Works Array support i i YYY a ri Samples y Incubation of Sample 1 2 hrs C Y with arrayed antibody jaha a e Cocktail of a oer 8 K AA Incubation with Y Y Biotinylated Ab ae Labeled 00 o o 4 streptavidin iq Incubation with 1 hrs yY labeled Streptavidin Detection of signals Data analysis l F and graph E RayBio G Series Glass Chip Layout BO E E Antibody j R E Array rray Bo E E L E g Bank gt _ L Blanx gt C O O O O OO O O Barco III Barcode li 4 arrays in one glass chip 8 arrays in one glass chip RayBio Human Acute Kidney Injury Antibody Array G Series 1 Ill Helpful Tips and General Considerations A Preparation and Storage of Samples 1 2 General Considerations e Freeze samples as soon as possible after collection e Avoid multiple freeze thaw cycles If possible sub aliquot your samples prior to initial storage e Spin samples hard 5 10 minutes at 10K to 15K RPM immediately prior to incubation
24. s using G Series Arrays 16 C Background Subtraction 17 D Normalization of Array Data 17 E Threshold of Significance 18 VI Antibody Array Map cccccecscesecssssessnsnnnnnnnee 19 VII Troubleshooting Guide cccccccccccccccccccsecccesseecseeeeeiees 20 VIIL Selected References 22 RayBio Cytokine Antibody Arrays are patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Human Acute Kidney Injury Antibody Array G Series 1 Introduction New techniques such as cDNA microarrays have enabled us to analyze global gene expression 13 However almost all cell functions are executed by proteins which cannot be studied simply through DNA and RNA techniques Experimental analysis clearly shows disparity can exist between the relative expression levels of MRNA and their corresponding proteins Therefore analysis of the proteomic profile is critical RayBiotech The Protein Array Pioneer Company introduced the first protein arrays to the market in 2001 and continues to lead in the development of innovative protein array technologies such as the RayBio Human Acute Kidney Injury Antibody Array Acute kidney injury is a common complication among ambulatory and hospitalized patients It is a rapidly progressive illness that independently predicts excess morbidity and mortality It is critical to early detect acute kidney injury and distinguish it from prerenal azotemia and chr
25. tages of infection by solid phase protein array J Neruovirol 2009 15 5 6 390 400 14 Pannebaker C Chandler HL Nichols JJ Tear proteomics in keratoconus Mo Vision 2010 16 1949 1957 23 RayBio Human Acute Kidney Injury Antibody Array G Series 1 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 Adiposin 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 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
26. ubation 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 Randomly scattered high intensity spots Dust or other particulates Dry slides in laminar flow hood and or use clean containers and powder free gloves 20 RayBio Human Acute Kidney Injury Antibody Array G Series 1 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 II and repeat Steps 12 21 Spin down reagents before diluting and mix well Other Tips Rescan at higher laser power or signal gain setting Repeat using higher sample concentration and or incubate wi sample O N at 4 C Increase concentration of and or length of incubation with Biotin conjugated Anti Cytokine add l large volume wash following Biotin Ab incubation Review proper storage conditions for kit components 21 RayBio Human Acute Kidney Injury Antibody Array G Series 1 Ill Selected References Citing RayBio Human G Series Arrays
27. ucts including customized products Just send your samples and we will send you the results Custom Services Customized Antibody and Protein Arrays Customized Phosphorylation Arrays Peptide synthesis Peptide arrays Recombinant protein and antibody production ELISA EIA Assay development Biostatistical amp Bioinformatic Analysis 0 Peptoid Synthesis amp Library Screening A OO Epa Technology Transfer Program Have you developed technologies or reagents of interest to the scientific and research community RayBiotech can help you commercialize your technologies reagents and your dreams 25 RayBio Human Acute Kidney Injury Antibody Array G Series 1 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 HiLyt
28. y 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 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 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 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 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 12 RayBio Human Acute Kidney Injury Antibody Array G Series 1 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 Il 11 Remove assembled glass chip from

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