Please read manual carefully before starting
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1. C or 80 C e We do not recommend comparing results between serum and plasma samples or between plasma prepared using different anticoagulants RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 6 e For most applications you may test plasma samples prepared using any anticoagulant i e Heparin EDTA or Citrate However EDTA prepared plasma may interfere with detection of MMPs and other metal binding proteins e If possible avoid testing hemolyzed serum or plasma as these samples may generate anomalous cytokine expression patterns and or high background signals B Handling Array Membranes e Array membranes are fragile when dry Handle with care Wet or dry grasp membranes by the edges using forceps Do not allow membranes to dry out during experiments The printed side of each membrane is denoted by a dash mark or array number in the upper left corner C Incubations and Washes e All washes and incubations in the standard protocol can be performed using the 8 well tray provided in the kit e Place the cover on 8 well trays with lid to avoid drying particularly during extended incubation or wash steps e During each incubation be sure to completely cover the membranes with sample or reagent e During incubation steps avoid foaming and be sure to remove all bubbles from the membrane surface e Perform all incubation and wash steps under gentle rotation or rocking motion 0 5 to 1 cycles second2. Cytokines to each membrane Incubate at RT for 1 2 hours 7 Decant or aspirate Anti Cytokine reagent and repeat washes as described in steps 4 and 5 above 8 Incubate at RT for 2 hours with 1 mL of 1X Streptavidin HRP 9 Wash membranes as directed in steps 4 and 5 10 Proceed with Detection protocol below or store membranes between plastic sheets provided in kit as directed in Steps 20 amp 21 below C Chemiluminescence Detection NOTE Do not allow membranes to dry out during detection Detection of chemiluminescence should be started within 5 minutes after removing Detection Buffers and must be completed within 20 minutes 11 Place a plastic sheet provided in the kit on your benchtop 12 Place one or more array membranes protein side up see Section III B on the plastic sheet Drain excess liquid by touching one edge to blotting paper or tissue paper RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 10 13 Into a single clean tube add equal volumes of Detection Buffer C and Detection Buffer D immediately prior to detection Mix well Add 250 uL of each buffer per membrane to be detected e g for 4 membranes combine 1 mL of each detection buffer 14 Pipette the mixed Detection Buffers onto each membrane Place another plastic sheet on top starting at one end and rolling the flexible plastic across the surface to the opposite end During this process ensure that the detection mixt
3. encephalopathy PNAS 2004 101 18 7070 7075 Tang X Marciano DL Leeman SE Amar S LPS induces the interaction of a transcription factor LPS induced TNF alpha factor and STAT6 B with effects on multiple cytokines PNAS 2005 102 14 5132 5137 Wang F X Xu Y Sullivan J et al IL 7 is a potent and proviral strain specific inducer of latent HIV 1 cellular reservoirs of infected individuals on virally suppressive HAART J Clin Invest 2005 155 128 137 De Ceuninck F Marcheteau E Berger S et al Assessment of Some Tools for the Characterization of the Human Osteo arthritic Cartilage Proteome J Biomol Tech 2005 16 256 265 RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 17 11 Boucharaba A Serre C M Guglielmi J et al The type 1 lysophosphatidic acid receptor is a target for therapy in bone metastases PNAS 2006 103 25 9643 9648 12 Matsunaga K Yanagisawa S Ichikawa T et al Airway cytokine expression measured by means of protein array in exhaled breath condensate Correlation with physiologic properties in asthmatic patients J Allergy Clin Immunol 2006 188 84 90 13 Vargas DL Nascimbene C Chitra Krishnan C et al Neuroglial activation and neuroinflammation in the brain of patients with autism Ann Neurol 2005 57 67 81 14 Miyoshi N Oubrahim H Chock PB Stadtman ER Age dependent cell death and the role of ATP in hydrogen peroxide induced apoptosis and necrosis PNAS 2006 1
4. primary organ responsible for the excretion of medications and their biotransformation products from the body Rats are widely used for probing pharmacokinetic pharmacodynamic PK PD relationships for medications in addition rats have been demonstrated to be a useful model for evaluating mechanisms of kidney toxicity In recent years numerous molecules have been described and investigated as candidate biomarkers of kidney injury The United States Food and Drug Administration FDA has taken an active role in developing a process for qualification of biomarkers that would potentially improve the drug development and regulatory review process In the gentamicin induced rat model of acute kidney RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol injury based on histopathology necrosis or apoptosis scoring kidney injury molecule 1 KIM 1 was the best biomarker of overall renal injury Traditionally urine proteins or cytokines are detected by using ELISA However RayBio Rat Acute Kidney Injury Antibody Array C Series can detect 7 protein biomarkers simultaneously with a small amount of sample It is a great tool in the acute kidney injury research areas and drug discovery area to hasten drug development 1 RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol Tang X Marciano DL Leeman SE Amar S LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT
5. 03 6 1727 1731 15 Coppinger JA O Connor R Wynne K et al Moderation of the platelet releasate response by aspirin Blood 2007 109 4786 4792 16 Cortez DM Feldman MD Mummidi S et al IL 17 stimulates MMP 1 expression in primary human cardiac fibroblasts via p38 MAPK and ERK1 2 dependent C EBP beta NF kB and AP 1 activation Am J Physiol Heart Circ Physiol 2007 293 H3356 H3365 17 Walt DR Blicharz TM Hayman RB Rissin DM et al Microsensor Arrays for Saliva Diagnostics Ann NY Acad Sci 2007 1098 389 400 RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 18 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 CNTF GITR IL 18 BPa MIP 15 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 a B y 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 TG
6. 6 B with effects on multiple cytokines PNAS 2005 102 14 5132 5137 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 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 50 2 91 106 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 Bonventre JV Weinberg JM Recent advances in the pathophysiology of ischemic acute renal failure Am Soc Nephrol 2003 14 2199 210 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 Goodsaid F Frueh F Biomarker qualification pilot process at the US Food and Drug Administration AAPS J 2007 9 E105 E108 Goodsaid FM Frueh FW Mattes W Strategic paths for biomarker qualification Toxicology 2008 245 219 223 9 Rodney L Rouse Jun Zhang Sharron R Stewart Barry A Rosenzweig Parvaneh Espandiari and Nakissa K Sadrieh3 Comparative profile of commercial
7. Acute Kidney Injury Antibody Array 1 C Series Protocol 20 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 Oe eo aN SS 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 Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 21 RayBio is the registered trademark of RayBiotech Inc The RayBio C Series Cytokine Antibody Array is 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 cl
8. Additional Materials Required e Small plastic boxes or containers Pipettors pipet tips and other common lab consumables Orbital shaker or oscillating rocker Saran Wrap or similar plastic film A chemiluminescent blot documentation system Such as UVP s ChemiDoc It or EpiChem II Benchtop Darkroom X ray Film and a suitable film processor or other chemiluminescent detection system RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol D How It Works Array support YYYYY a Pe Samples Incubation of Sample with arrayed antibody supports 1 2 hrs a AKK Labeled e streptavidin iC Incubation with 1hrs Incubation with Biotinylated Ab 1 2 hrs labeled Streptavidin KK t se 7 Detection of n signals Data analysis o and graph Ill Helpful Tips and General Considerations A Preparation and Storage of Samples 1 General Considerations RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 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 with each sample If spot intensities are weak increase sample
9. Antibody Arrays Quantibody Multiplex ELISA Arrays L Series Biotin Label based Antibody Arrays Phosphorylation Antibody Arrays o Receptor Tyrosine Kinases o EGFR and ErbB family site specific phosphorylation Over 700 different ELISA kits EIA kits Cell based phosphorylation assay Over 10 000 different Antibodies Recombinant proteins Peptide Recombinant antibodies OO0O000 0 7 Protocol for RayBio Rat Acute Kidney Injury Antibody A Array 1 C Series TABLE OF CONTENTS l HO CIC O esccsnecisnan a 1 Il Product Information sssiessiisiiieiissriserrsrreernen 3 A Storage Recommendations 3 B Materials Provide cccccccccccccccccccseecsesseesteneeseeen 4 C Additional Materials Required 4 D HOW lt WOrKS oon eecsssscesssssssecssssssseeesssstetesesssstnesesssteneeenseene 5 Ill Helpful Tips and General Consideratione 5 A Preparation and Storage of Samples 5 B Handling Array Membranes 7 C Incubations and Washes 7 D Chemiluminescence Detection 8 IV Proto COl 8 A Preparation and Storage of Reagent 8 B Blocking and Incubations 9 C Chemiluminescence Detection 00 10 V Interpretation of Results 12 VI Antibody Array Map 15 VII Troubleshooting Guide ccccccccccsccesccscsssssessesseeeeee 16 VII Selected References 17 RayBio Cytokine Antibody Arrays are patent pending technology RayBio is the trademark of RayBiote
10. FB3 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 81 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 CA15 3 Ferritin IL 10 LH 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 88 1 GDNF IL 17R MIP 1B SDF 1B RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 19 RayBio Rat
11. ODG INGAL TIMP 1 18 26 DM 11 12 13 14 45 16 17 18 19 20 2 22 23 24 nee SEETI TT 5 28 O08 0 G05 M roan RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 15 VII Troubleshooting guide Problem Cause Recommendation No signal for any spots including Positive Controls Global detection failure Repeat incubation with HRP Streptavidin and Detection Buffers Weak or no signals antigen specific spots Sample is too dilute Improper dilution of HRP Streptavidin Repeat experiment using higher sample concentration Tube may contain precipitants Repeat detection mix 1000X HRP Streptavidin well before diluting reagent Waiting too long to detect chemiluminescent signals Repeat detection making sure to complete this process within 20 min Other Tips Incubate with sample O N at 4 C Increase concentration of HRP Streptavidin Increase concentration of Biotin conjugated Anti Cytokine Extend exposure time may go overnight Uneven signal or background Bubbles present on membrane during incubations Be sure to completely remove all bubbles from membrane surface Membranes were not evenly covered during washes incubations or allowed to dry out Completely cover membranes with solution use a rocker or shaker during washes and incubations High background signals Overexposure Decrease exposure time Sample is t
12. RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Patent Pending Technology User Manual RayBio Rat Acute Kidney Injury Antibody Array C Series Cat AAR AKI 1 4 RayBio Rat Acute Kidney Injury Antibody Array C Series Cat AAR AKI 1 8 RayBio Rat Cytokine Antibody Array Service Cat AAR SERV Please read manual carefully before starting experiment Ris RayBiotech Inc 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 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 1000 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable and cost effective Our product offerings include 1 2 OL Protein antigen Arrays Cytokine Antibody Arrays Human Mouse Rat and Porcine o C Series Membrane chemiluminescence detection o 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 Inflammation Antibody Arrays MMP Antibody Arrays o Obesity
13. 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 as 1X concentration no reconstitution or dilution is required Store at 20 C or 80 C when not in use 2 Wash Buffers and II ltem 0103004 W are supplied as 20X a For each membrane dilute 1 mL of Wash Buffer with deionized water to a final volume of 20 mL b For each membrane dilute 1 mL of Wash Buffer II with deionized water to a final volume of 20 mL c 1X Wash Buffers can be stored at 4 C for up to 1 month 20X Wash Buffers can be stored 4 C for up to 3 months 3 Biotin Conjugated Anti Cytokines are supplied at 2000X concentration as a small liquid bead typically 2 5 uL RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 8 Note Spin down the tube prior to reconstitution as the concentrated liquid bead may have moved to the top of the tube during handling a Add 100 uL 1X Blocking Buffer to the tube containing 2000X Biotin Conjugated Anti Cytokines b Mix well and quantitatively transfer stock reagent to larger tube containing 1900 uL of 1X Blocking Buffer c 1X Biotin Conjugated Anti Cytokines may be stored for 2 3 days at 4 C 4 Streptavidin HRP is supplied as 1000X concentration a Mix the tube containing 1 000X Streptavidin HRP well before use as precipitates may form during storage b Ad
14. aim relating to products shall be limited to replacement or refund of the purchase price X Omat is the trademark of The Kodak Company ChemiDoc It is the registered trademark of UVP This product is for research use only 2011 RayBiotech Inc RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 22
15. ch Inc RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 0 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 Rat 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 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 insult The kidney is the
16. concentration in subsequent experiments If background or spot intensities are too strong decrease sample concentration in subsequent experiments e 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 the 1X Blocking Buffer provided in this kit For all sample types final sample volume 1 0 1 2 mL per membrane e Urine 2 fold to 5 fold dilution Note If the sample volume is less than 200ul the membrane and sample may be sealed in a small plastic bag to increase sample membrane coverage Expel all air bubbles prior to sealing bag Note The Ra yBio Rat Acute Kidney Injury Antibody Array is intended for use with Rat 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 200ul 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 precipitates 4 Preparing Serum Plasma e Prepare samples according to established protocols or collection tube manufacturer s instructions Sub aliquot into plastic tubes Store at 20
17. d 2 uL of 1000X Streptavidin HRP to 1998 uL of 1X Blocking Buffer c This working dilution can be stored for 3 5 days at 4 C 5 Detection Buffers C amp D are supplied as 1X solutions that are intended to be mixed in a 1 1 ratio immediately prior use Detection reagents may be stored at 4 C for up to 3 months B Blocking and Incubations NOTE Please prepare all reagents immediately prior to use as described above Section IV A and carefully read tips on Sample Preparation Section III A and Incubations and Washes Section III C before proceeding 1 Place each membrane printed side up see Section III B into the 8 well tray provided in the kit 2 Block membranes by incubating with 2 mL 1X Blocking Buffer at room temperature RT for 30 min 3 Decant Blocking Buffer and incubate membranes with 1 mL of sample at RT for 1 2 h RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 9 4 Aspirate samples from membranes Wash 3 times 5 min per wash with 2 mL Wash Buffer at RT Use fresh buffer for each wash OPTIONAL Large Volume Wash After Step 4 place membranes into clean container s Add 20 30 mL of Wash Buffer per membrane and wash at RT with gentle shaking or rocking for 30 45 min Return membranes to the 8 well tray Then proceed to Step 5 5 Wash 2 times 5 min per wash with 2 mL of 1X Wash Buffer ll each at RT Use fresh wash buffer each time 6 Add 1 mL of 1X Biotin conjugated Anti
18. e 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 antigen specific spot intensities e If you perform overnight sample incubations we recommend adding the optional Large Volume Wash described in Step 4 to minimize background signals e Overnight blocking and wash steps are useful for reducing background signal intensities Wash steps may be repeated even with completed membranes to reduce background signals Wash with Wash Buffer Il followed by repeating incubation with Streptavidin HRP and chemiluminescent RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 7 detection may greatly improve signal to noise ratios in your developed array images D Chemiluminescence Detection e We strongly recommend using multiple exposures to obtain optimum images Begin by exposing the membranes for 40 seconds Then re expose the film accordingly e f the signals are too strong or background is too high reduce exposure time e g 5 30 seconds e Ifthe signals are weak increase exposure time e Gel blot documentation systems that use CCD cameras to detect chemiluminescence are ideal for imaging RayBio array membranes They can easily be programmed to take multiple exposures and the dynamic range of these detectors tends to be 2 3 orders of magnitude greater than X ray film IV Protocol A Preparation and
19. ly available urinary biomarkers in preclinical drug induced kidney injury and recovery in rats Kidney International 2011 79 1186 1197 Il Product Information A Storage Recommendations For best results store the entire kit at 20 C or 80 C upon arrival If stored frozen we recommend using the kit within 6 months which is the duration of the product warranty period Once thawed store array membranes 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 Array kits are robust and will retain full activity even if stored for up to 24 hours at room temperature RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol B Materials Provided 7 AAR AAR Item Description AKI 1 4 AKI 1 8 2 RayBio Rat AKI Antibody 1or2 PSE Array Membranes paks 2 paks paw 1 Biotin Conjugated Anti aaa a Cytokines 2ea 4ea 0103004 H 1 000X HRP Conjugated Streptavidin lea lea 0103004 B 1X Blocking Buffer 25ml 50ml 0103004 W T 20X Wash Buffer I T 10ml 20ml 0103004 W T 20X Wash Buffer II T 10m 20ml 0103004 D T Detection Buffer C T 15m 25ml 0103004 D T Detection Buffer D T 15m 25ml 0103004 T 8 Well Plastic Tray 1 1 Other Kit Components 8 well Tray Plastic sheets Manual Array Template Packing list Packs contains 2 or 4 arrays each t Wash Buffers and Detection Buffers are sold as Sets Y 4or8 C
20. ol and or Blank spots will be included Blank spots are literally blank nothing has been printed there Negative Control spots are printed with the same buffer used to dilute antibodies printed on the array Thus the signal intensities of the Negative Controls represent the RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 13 background plus non specific binding to the printed spots We recommend subtracting the mean of 4 or more Negative Control spots for background correction Normalization of Array Data The amount of biotin conjugated IgG protein printed for each Positive Control spot is consistent from array to array As such the intensity of these Positive Control signals can be used to normalize signal responses for comparison of results across multiple arrays much like housekeeping genes and proteins are used to normalize results of PCR gels and Western Blots respectively To normalize array data one 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 first column on the left of 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 density of Positive Control spots on reference array P y mean signal density of Positive Control spots on Array y X y mean
21. on systems are usually sold as a package with compatible densitometry software To obtain densitometry data from an X ray film one must first scan the film to obtain a digitized image using an ordinary office scanner with resolution of 300 dpi or greater Any densitometry software should be sufficient to obtain spot signal densities from your scanned images One such software program ImageJ is available for free from the NIH for more info visit http rsbweb nih gov ij We suggest using the following guidelines when extracting densitometry data from our array images For each array membrane identify a single exposure that the exhibits low background signal intensity and strong Positive Control signals that do not bleed into one another Exposure times do not need to be identical for each array but Positive Control signals on each image should have similar intensities e Measure the density of each spot using a circle that is roughly the size of one of the largest spots Be sure to use the same circle area and shape for measuring the signal densities on every array for which you wish to compare the results e For each spot use the summed signal density across the entire circle i e total signal density per unit area Before analysis subtract the background from raw densitometry data and normalize the signal intensities to the Positive Controls Background Subtraction On each array several Negative Contr
22. oo concentrated Repeat experiment using more dilute sample NOTE To reduce background on completed membrane wash O N 4 C in Wash Buffer II then re incubate with HRP Streptavidin and repeat detection RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 16 VIII Selected References Featuring RayBio C Series Arrays 1 10 Ray S Britschgi M Herbert C et al Classification and prediction of clinical Alzheimer s diagnosis based on plasma signaling proteins Nature Med 2007 13 11 1359 1362 McAllister SS Gifford AM Greiner AL et al Systemic Endocrine Instigation of Indolent Tumor Growth Requires Osteopontin Cell 2008 133 994 1005 Acosta JC O Loghlen A Banito A et al Chemokine Signaling via the CXCR2 Receptor Reinforces Senescence Cell 2008 133 1006 1018 Brown JM Boysen MS Chung S et al Conjugated Linoleic Acid Induces Human Adipocyte Delipidation Autocrine Paracrine Signaling by Adipokines J Biol Chem 2004 279 25 26735 26747 Celis JE Moreira JMA Gromova I et al Towards discovery driven translational research in breast cancer FEBS J 2004 272 2 15 Lin PW Simon PO Gewirtz AT et al Paneth Cell Cryptdins Act in Vitro as Apical Paracrine Regulators of the Innate Inflammatory Response J Biol Chem 2004 279 19 19902 19907 Xu Y Kulkosky J Acheampong E et al HIV 1 mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced
23. rrays Sample 1 Sample 2 Control ee s eo s o ao ee z o ae va ee G ia a EJ r amp E E a e amp D bd e a amp a The preceding figure presents typical images obtained with RayBio Human Cytokine Antibody Array These membranes were probed with conditioned media from two different cell lines Membranes were exposed to Kodak X Omat film at RT for 1 min Note the strong signals of the Positive Control spots provided by biotin conjugated IgG printed directly onto the array membrane in the upper left and lower right corners These Positive Control spots are useful for proper orientation of the array image The signal intensity for each antigen specific antibody spot is proportional to the relative concentration of the antigen in that sample Comparison of signal intensities for individual antigen specific antibody spots between and among array images can be used to determine relative differences in expression levels of each analyte sample to sample or group to group Obtaining Densitometry Data Visual comparison of array images may be sufficient to see differences in relative protein expression However most researchers will want to perform numerical comparisons of the signal intensities or more precisely signal densities using 2 D RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 12 densitometry Gel Blot documentation systems and other chemiluminescent or phosphorescent detecti
24. signal density for spot X on Array for sample y X Ny normalized signal intensity for spot X on Array y After background subtraction and normalization you can compare signal intensities analyte by analyte among or between samples or groups to determine relative differences in cytokine expression The RayBio Analysis Tool software is available for use with data obtained using RayBio Cytokine Antibody Arrays Copy and paste your signal intensity data into the Aligning Data worksheet and it will compile and organize your data as well as automatically subtracting background signals and normalizing to RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 14 the Positive Controls To order the Analysis Tool please contact us at 1 770 729 2992 or information info raybiotech com for more Vi RayBio C Series Rat Acute Kidney Injury Antibody Array 1 Maps Detects 7 cytokines in one experiment A B C D E F G H 1 POS POS NEG NEG CystatinC FABP1 KIM 1 MCP 1 2 POS POS NEG NEG CystatinC FABP1 KIM 1 MCP 1 3 NGAL TIMP 1 VEGF NEG NEG NEG NEG POS 4 NGAL TIMP 1 VEGF NEG NEG NEG NEG POS Abbreviations POS Positive Control NEG Negative Control L FABP Liver Fatty Acid Binding Protein KIM 1 Kidney Injury Molecule 1 NGAL Neutrophil Gelatinase Associated Lipocalin Lipocalin 2 All others use standard abbreviations 1 2 9 10 24 32 OOAD
25. ure completely covers each membrane and gently smooth out any air bubbles Avoid sliding the plastic sheet along the membranes printed surfaces 15 Incubate at RT for 2 minutes 16 Remove top plastic sheet and aspirate excess liquid see Step 12 17 Gently replace the membranes protein side up on the bottom plastic sheet and replace the top plastic sheet see Step 14 Gently smooth out any air bubbles on the membrane surfaces 18 Detect signals using a chemiluminescence imaging system or expose the array membranes to x ray film we recommend Kodak s X Omat AR film and develop the film See tips for obtaining array images in Section III D 19 For each array use multiple exposures to obtain an image with low background and strong Positive Control signals that do not bleed into one another Typical exposure times are 10 seconds to 2 min 20 When you finish your last exposure remove the top plastic sheet Gently rinse membranes and plastic sheets with Wash Buffer Il Remove excess wash buffer as described in Step 14 and replace the membranes between the plastic sheets RayBio Rat Acute Kidney Injury Antibody Array 1 C Series Protocol 11 21 Wrap the sheets in Saran Wrap and store the membranes at 20 C to 80 C Or store membranes for up to 5 days at 4 C in Wash Buffer II Cover the container to avoid evaporation V Interpretation of Results Typical results obtained with RayBio C Series Antibody A
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