G Series
Contents
1. 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 Protocol 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 19 Similar signal intensities for POS1 2 3 High background signals Improper laser power and or PMT setting Incomplete washes Sample concentration is too high Repeat scan using higher and or lower laser power or PMT settings Carefully follow wash protocols and or increase wash times 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 Randomly scattered high intensity 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 precipita2. ccccccccccccccneeen 15 V Interpretation Of RESUItS eens 15 A Explanation of Control Spots 15 B Typical Results using G Series Arrays 15 C Background Subtraction 16 D Normalization of Array Data 16 E Threshold of Significance 18 VI Antibody Array Map eee cecsssssssssssnnnnnnnses 19 VII Troubleshooting Guide cccccccccccccccccccccccsssccsssssseesseeeetees 20 VII 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 Protocol Introduction New techniques such as cDNA microarrays have enabled us to analyze global gene expression However almost all cell functions are executed by proteins which cannot be studied simply through DNA and RNA techniques Experimental analysis clearly shows disparity can exist between the relative expression levels of mRNA and their corresponding proteins Therefore analysis of the proteomic profile is critical 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
3. al Ne sseria gonorrhoeae Activates the Proteinase Cathepsin B to Mediate the Signaling Activities of the NLRP3 and ASC Containing Inflammasome J mmuno l 2009 182 6460 6469 Pukstadad BS Ryana L Floa TH JStenvika 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 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 Mo Cell Proteom 201 9 1085 1099 Streblow DN Dumortier J AMoses 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 Mo Vision 2010 16 1949 1957 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 24 Customized RayBio Cytokine Antibody Arrays Select your cytokines
4. 26 RayBio Cytokine Antibody Arrays are a 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 GenePix is a registered trademark of Molecular Devices Inc This product is for research use only 2011 RayBiotech Inc RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 27
5. 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 Anaspec Inc Wash Buffers are sold as sets X 4 or 8 based on the number of printed sub arrays on the chip RayBio Human Acute Kidney Injury Antibody Array G Series Protocol C Additional Materials Required Small plastic boxes or containers Pipettors pipette tips and other common lab consumables Orbital shaker or oscillating rocker Aluminum foil Wash bottle Gene microarray scanner or similar laser fluorescence scanner D How It Works Array support i YYY a a Samples Incubation of Sample Y with arrayed antibody Wry supports 1 2 hrs Cocktail of K x K x 4 Biotin Ab KK Incubation with Y Y Biotinylated Ab ne streptavidin E T Incubation with Yoy labeled Streptavidin 1hrs s a 5 Detection of signals Data analysis iS and graph RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 7 E RayBio G Series Glass Chip Layout BL anitody Hi C gt E BL Bank gt _ L LI LI Barcode 1 1 II Antibody Array Blank gt Barcode 8 arrays in one glass chip Ill Helpful Tips and General Considerations A Preparation and Storage of Samples 1 General Considerations Freeze samples as soon as possible after collection Avoid multiple freeze thaw cycles If possible sub aliquot your samples prior
6. frequency 532 nm For tips on scanning visit our Website http www raybiotech com Tech Support Scanning Tips pdf NOTE If you do not have a laser scanner RayBiotech offers scanning and data extraction services for a nominal fee Also using alternate protocols RayBio G Series arrays are compatible with Li Cor s Odyssey and Gentel BioScience s APIX scanners For more information contact RayBiotech 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 Steptavidin Fluor Negative control signal intensities are usually very close to background signals in each sub array B Typical results obtained with RayBio G Series Antibody Arrays The following figure shows typical results obtained using RayBio G Series Antibody Arrays The images were captured using a GenePix 4000B scanner Ray
7. morbidity and mortality It is critical to early detect acute kidney injury and distinguish it from prerenal azotemia and chronic 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 insulkt 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 Lipocalin NGAL Kidney Injury Molecule 1 KIM 1 Cystatin C Liver Fatty acid Binding Protein L FABP TEES Chemoattractant Protein 1 MCP 1 and Trefoil Factor 3 TFF3 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 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 to
8. to initial storage Spin samples hard 5 10 minutes at 10K to 15K RPM immediately prior to incubation of samples with array sample concentrations may need determined empirically based on the signal intensities of spots and background signals obtained Most samples will not need to be concentrated concentration is required we recommend using a spin column concentrator with a chilled centrifuge 2 Recommended Sample Volumes and Dilution Factors NOTE All sample dilutions should be made using 1X Blocking Buffer For all sample types final sample volume 50 100 uL per sub array RayBio Human Acute Kidney Injury Antibody Array G Series Protocol Optimal IIS IIB ES LJ LJ HIM e Urine 2 fold to 5 fold dilution Note The RayBio Acute Kidney Injury Antibody Array is intended for use with human urine samples However if you wish you may test other sample types as follows e Serum amp Plasma 2 fold to 5 fold dilution 3 Preparing Urine e Prepare 500 uL aliquots and store at 20 C or 80 C as soon as possible after collecting urine samples e Addition of protease inhibitors is not required e Immediately prior to sample incubation Step 3 of protocol spin samples at 1000 rpm for 10 minutes to remove particulates and precipitants 4 Preparing Serum Plasma e Prepare samples according to established protocols or collection tube manufacturer s instructions Sub aliquot into plastic tu
9. 0X concentrate with deionized H20 to a final volume of 100 mL each of Wash Buffer amp Wash Buffer II b Wash Buffer reagents at 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 as a small liquid bead typically 2 5 uL of highly concentrated antibodies 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 4 Streptavidin Fluor is supplied as 1500X 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 i e 1 5 mL d This working dilution can be stored for 3 5 days at 4 C RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 11 B Blocking and Incubations NOTE Please carefully read Section Ill of this manual before proceeding NOTE Prepare all reagents immediately prior to use as described above Section IV A before proceed
10. 5 3 Ferritin IL 10 PECAM 1 TREM 1 CA19 9 FGF 4 IL 10 Ra Lymphotactin PIGF TSH CA IX FGF 6 IL 10 RB LYVE 1 PF4 TSLP Cardiotrophin 1 FGF 6 IL 11 Marapsin Procalcitonin Ubiquitin Cathepsin S FGF 7 IL 12 MCP 1 Prolactin uPAR CCL14a FGF 9 IL 12 p40 MCP 2 PSA free VCAM 1 CCL21 Fit 3 Ligand IL 12 p70 MCP 3 PSA total VE Cadherin CCL 28 FLRG IL 13 MCP 4 RAGE VEGF CD14 Follistatin IL 13 Ra 2 M CSF RANK VEGF R2 CD23 Fractalkine IL 13 RI M CSF R RANTES VEGF R3 CD30 FSH IL 15 MDC Resistin VEGF C CD40 Furin IL 16 MICA S 100b VEGF D CD40 Ligand Galectin 7 IL 17 MICB SAA XEDAR CD80 GCP 2 IL 17B MIF SCF CEA G CSF IL 17C MIG SCF R CEACAM 1 GDF 15 IL 17F MIP 1a SDF 1 CK b 8 1 GDNF IL 17R MIP 1B SDF 1B RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 25 Testing Services RayBiotech offers full testing services using any of our Array ELISA or EIA products including customized products Just send your samples and we will send you the results Custom Services Customized Antibody and Protein Arrays Customized Phosphorylation Arrays Peptide synthesis Peptide arrays Recombinant protein and antibody production ELISA EIA Assay development DSTO OnE 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 dream RayBio Human Acute Kidney Injury Antibody Array G Series Protocol
11. Bio Human Acute Kidney Injury Antibody Array G Series Protocol 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 BF Array VI Array VI Array VIN Patient Serum 1 Patient Serum 2 Patient Serum 3 Negative Control Note the 6 strong signals of the Positive Control spots in the upper left corner These spots 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 automatically measure the local background around each spot As RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 17 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
12. RayBio Human Acute Kidney Injury Antibody Array 1 G Series Patent Pending Technology User Manual 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 TH17 G1 8 RayBio Human Acute Kidney Injury Antibody Array G Series Testing Services Cat AAH SERV G Please read manual carefully before starting experiment RayBiotech Inc a the protein array pioneer company We provide you with excellent Protein Array systems and services Tel Toll Free 1 888 494 8555 or 1 770 729 2992 Fax 1 770 206 2393 Website www raybiotech com Email info raybiotech com RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 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 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 Ar
13. atives 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 Neuromusc 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 Clin Immunol 2009 doi 10 1016 j clim 2009 07 001 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 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 23 10 11 12 13 Duncan JA Gao X Huang MT H O Connor BP Thomas CE et
14. bes Store at 20 C or 80 C We do not recommend comparing results between serum and plasma samples or between plasma prepared using different anticoagulants You may test plasma samples prepared using any anticoagulant i e Heparin EDTA or Citrate However EDTA prepared plasma may interfere with optimal detection of MMPs and other metal binding proteins If possible avoid testing hemolyzed serum or plasma as these samples may generate anomalous cytokine expression patterns and or high background signals 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 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol Avoid breaking glass chip when removing the chamber assembly C Incubations and Washes Cover incubation chamber with adhesive film included in kit to prevent evaporation particularly during incubation or wash steps gt 2 h or with liquid volumes lt 100 uL per well Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycle s Wash steps in Wash Buffer II and all incubation steps may be performed overnight at 4 C Overnight sample incubations are the most effective at increasing sample spot intensities Avoid cross contam
15. chip from the frame assembly Place the chip in 30 mL Centrifuge Tube provided in kit or slide staining jar Add enough 1X Wash Buffer to cover the whole slide about 20 mL and gently rock or shake at RT for 10 min Decant buffer and repeat wash as described in Step 16 this time using 1X Wash Buffer Il Decant buffer remove the glass chip from the tube then gently rinse the slide with distilled H2O using a plastic wash bottle Remove excess liquid from 30 mL Centrifuge Tube and place glass chip into the tube Centrifuge at 1 000 RPM for 3 minutes to remove water droplets Obtaining Fluorescent Signal Intensities 19 20 Remove chip 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 completely dry before scanning or storage You may proceed immediately to scanning Step 21 or you may store the slide at 20 C in the centrifuge tube provided or at RT and to scan at a later time Note Unlike most Cy3 fluors the HiLyte Plus Fluor 532 used in this kit is very stable at RT and resisiant to photobleaching on completed glass chips However please protect glass chips from strong light and temperatures above RT RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 15 21 Scan the glass chip with a laser scanner such as Axon GenePix using Cy3 or green channel excitation
16. 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 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 19 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 POS POS NEG NEG KIM 1 ALB OPN TFF3 2 POS POS NEG 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 P 10 CystatinC HGF MIF NGAL TIMP 1 VCAM 1 VEGF 6 IP 10 Cystatin C HGF MIF NGAL TIMP 1 VCAM 1 VEGF 7 NEG NEG NEG NEG NEG NEG NEG POS 8 NEG NEG NEG NEG NEG NEG NEG POS Abbreviations POS Positive Control NEG
17. ination of samples to neighboring wells To remove Wash Buffers and other reagents from chamber wells you may invert the Incubation Chamber Assembly to decant and aspirate the remaining liquid In Wash Steps 6 12 and 15 you may gently flush wells several times using a wash bottle filled with Wash Buffer I D 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 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 Item 0103004 B is supplied as 2X a For glass chips with 4 sub arrays each prepare at least 2 5 ml 1 25 mL 2X Blocking Buffer 1 25 mL deionized H20 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol b For glass chips with 8 sub arrays each prepare at least 3 0 ml 1 5 mL 2X Blocking Buffer 1 5 mL deionized H20 c If your samples require dilution prior to incubation with the sub arrays increase this volume accordingly d Store 1X and 2X Blocking Buffer at 20 C or 80 C when not in use 2 Wash Buffers and II Item 0103004 W are supplied as 20X a For each glass chip 4 or 8 sub arrays chip dilute 5 mL of 2
18. ing 1 Remove the package containing the Glass Chip Assembly from the freezer Place unopened package on the benchtop and allow the it 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 on pages 12 13 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 array surface NOTE Only add reagents or samples to wells printed with antibodies see diagram on page 7 Instructions for incubation chamber assembly G Series and Quantibody Arrays Incubation chamber gt Gasket normally attached to chamber Protective Cover can et be discarded Snap on sides RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 12 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 7 Carefully place slide at bottom of the chamber 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 proced
19. l 16 1886 1903 2005 8 Mishra J Dent C Tarabishi R Mitsnefes MM Ma Q Kelly C Ruff SM Zahedi K Shao M Bean 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 Protocol 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 Intl 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 Intl 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 J 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 injur
20. ner and replenish with 1X Wash Buffer Again submerge the entire glass chip assembly and wash 10 min at RT with gentle rocking or shaking Remove Glass Chip Assembly and invert to decant liquid Decant buffer from container and repeat Steps 8 amp 9 with 1X Wash Buffer II Invert Glass Chip Assembly to decant liquid then carefully aspirate wash buffer from wells touching only the corners with your pipette tip Add 70 uL of 1X Biotin conjugated Anti Cytokines to each sub array Cover Incubation Chamber with Adhesive Film Incubate at RT for 2 hours with gentle rocking or shaking Carefully aspirate Biotin conjugated Anti Cytokine reagent Wash as described in Step 7 above first with 1X Wash Buffer then with 1X Wash Buffer Il making sure to completely remove buffer between washes and after final wash Add 70 uL of 1X Streptavidin Fluor to each sub array Cover the incubation chamber with Adhesive film then cover entire assembly with aluminum foil to avoid exposure to light or RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 14 15 16 17 18 incubate in dark room Incubate at RT for 2 hours with gentle rocking or shaking Remove aluminum foil and Adhesive Film Carefully aspirate Streptavidin Fluor reagent Wash as described in Step 7 above first with 1X Wash Buffer then with 1X Wash Buffer II making sure to completely remove buffer between washes and after final wash Remove glass
21. of interest from the following list and we will produce the customized array for you For more information please visit our website www raybiotech com 4 1BB CNTF GITR IL 18 BPa MIP 16 SAA ACE 2 Cripto GITR Ligand IL 18 RB MIP 3a sgp130 Acrp30 CRP GM CSF IL 1ra MIP 3B Shh N Activin A CTACK GRO IL 2 MMP 1 Siglec 5 Adiposin CXCL16 GROa IL 2 RB MMP 10 Siglec 9 Adipsin DAN GH IL 2 Ry MMP 13 ST2 AgRP Decorin HB EGF IL 2 Ra MMP 2 sTNF RI ALCAM Dkk 1 HCC 4 IL 21R MMP 3 sTNF RII a Fetoprotein Dkk 3 hCG intact IL 22 MMP 7 TACE Amphiregulin Dkk 4 HGF IL 28A MMP 8 TARC Angiogenin DPPIV HVEM IL29 MMP 9 TECK Angiopoietin 1 DR6 1 309 IL 3 MPIF 1 TGFa Angiopoietin 2 Dtk ICAM 1 IL 31 MSPa TGFB1 Angiostatin E Cadherin ICAM 2 IL 4 NAP 2 TGFB2 ANGPTL4 EDA A2 ICAM 3 IL 5 NCAM 1 TGFB3 Axl EGF IFNy IL 5 Ra NGF R TPO B7 1 EGFR IGF 1 SR IL 6 Nidogen 1 Thyroglobulin BCAM EG VEGF IGFBG 1 IL 6 sR NrCAM Tie 1 BCMA ENA 78 IGFBP 2 IL 7 NRG1 1 Tie 2 BDNF Endoglin IGFBP 3 IL 8 NT 3 TIM 1 B2M Eotaxin IGFBP 4 IL 9 NT 4 TIMP 1 B IG H3 Eotaxin 2 IGFBP 6 Insulin OncostatinM TIMP 2 bFGF Eotaxin 3 IGF I IP 10 Osteopontin TIMP 4 BLC Ep CAM IGF I SR I TAC OPG TNFa BMP 4 ErbB2 IGF II LAP PAI I TNFB BMP 5 ErbB3 IL 1a Leptin PARC TNFRSF21 BMP 6 EPOR IL 1B Leptin R PDGF Ra TNFRSF6 BMP 7 E Selectin IL 1 R Il LIF PDGF RB TRAIL R2 B NGF Fas IL 1 R4 ST2 LIGHT PDGF AA TRAIL R3 BTC Fas Ligand IL 1 RI LIMPII PDGF AB TRAIL R4 CA125 Fer RIIB C IL 1 sRI L Selectin PDGF BB Trappin 2 CA1
22. ol in the acute kidney injury research areas including drug toxicity monitoring kidney transplantation rejection reaction 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 Gia 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 Am Soc Nephro
23. rays Growth Factor Antibody Arrays Inflammation Antibody Arrays MMP Antibody Arrays o Obesity Antibody Arrays Quantibody Multiplex ELISA Arrays RayBio L Series Biotin Label based Antibody Arrays RayBio Phosphorylation Antibody Arrays Receptor Tyrosine Kinases 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 OO0O000 0 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 1 TABLE OF CONTENTS LITO MUCHION eee ceccccccccseeestenesssssecsssssssnnesecccesssssnneeseeceecsssnssseees 3 HW Product INFOPMATION 0 cc ccc ccccccccsssseessssesssstecesseceestesesseseenees 5 A Storage Recommendations 0 00 cceee 5 B Materials Provided cccccccesesceeeteeeesnneeeees 6 C Additional Materials Required 7 D How lt WV ONS itstshsstticccdecteteses the ces crstecciettlnenst tt acioteacees 7 E RayBio G Series Glass Chip Layout 8 Ill Helpful Tips and General Considerations 8 A Preparation and Storage of Sampleg 8 B Handling Glass Chip c cceeeeeeeiee 9 C Incubations and Washes 9 D Data Extraction Tips sees 10 W Protocole 10 A Preparation and Storage of Reagents 10 B Blocking and Incubations 12 C Fluorescence Detection
24. 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 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 Threshold of significant difference in expression RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 18 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
25. tion 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 Dust or other particulates Repeat experiment using more dilute sample Dry slides in laminar flow hood and or use clean containers and powder free gloves RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 21 Weak or no signals antigen specific spots 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 19 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 addl l large volume wash following Biotin Ab incubation Review proper storage conditions for kit components RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 22 Ill Selected References Citing RayBio Human G Series Arrays 1 Kocaoemer A Kern S Kluter H Bieback K Human AB Serum and Thrombin Activated Platelet Rich Plasma Are Suitable Altern
26. ure with the other side 4 Decant Blocking Buffer then aspirate remaining liquid 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 1 to 2 hours with gentle rocking or shaking 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 RayBio Human Acute Kidney Injury Antibody Array G Series Protocol 13 7 Wash each array 3 times 2 min per wash 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 NOTE Try to prevent solution from flowing into neighboring wells 8 10 11 12 13 14 Obtain a clean container e g 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 Remove Glass Chip Assembly and invert it to decant liquid Decant buffer from contai
27. y Nat Biotechnol 2010 May 28 5 470 7 Il Product Information A Storage Recommendations For best results we recommend storing the entire kit at 20 C or 80 C upon arrival and using the kit within 6 months of receipt RayBiotech warranties this product for 6 months if stored in this manner Once thawed store glass chips and 2X 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 Protocol B Materials Provided AAH AAH _ TH17 TH17 Item Description G1 4 G1 8 RayBio Human Acute Kidney 1 chip with 1 chip with AAH AKI1 GX Injury Antibody Microarray Glass 4 Sub 8 Sub Chip arrays arrays 0103002 HAK Biotin Conjugated Anti Cytokines 1 ea 2ea 1 500X HiLyte Plus 532 ar Streptavidin Fluort Ka Ia 0103004 B 2X Blocking Buffer 10 mL 10 mL 0103004 W 20X Wash Buffer 30 mL 30 mL 0103004 W t 20X Wash Buffer II 30 mL 30 mL 0103004 L 2X Cell Lysis Buffer optional 10 mL 20 mL Other Kit Components Manual Adhesive Plastic Strips 30 mLCentrifuge 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
Download Pdf Manuals
Related Search