Human Cytokine Antibody Array C series 1000
Contents
1. Human Cytokine Antibody Array C Series 1000 Patent Pending Technology User Manual Revised February 4 2009 RayBio Human Cytokine Antibody Array C series 1000 Combination of Array 6 amp 7 Cat AAH CYT 1000 Please read manual carefully before starting experiment RayBiotech Inc We Provide You with Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com 4 RayBiotech Inc RayBio Human Cytokine Antibody Array C Series 1000 Protocol TABLE OF CONTENTS I InttoductlODl eoi iie ke 2 How It 4 Materials 5 Additional Materials 5 III Overview and General Considerations 6 A Preparation of 6 B Handling Array 6 C 1 aude ares ehe 6 IV Protocol hS KK KK KK KK KK KK KK 7 A Blocking and Incubation 7 B Detection iii landed ad 9 V Interpretation of 10 Troubleshooting 14 VII Reference ISU LL KK KK K 15 Cytokine Antibody Arrays are RayBiotech patent pending technology RayBio is the trad2. Takeda Uchimura Y et al Nature Med 2007 13 11 1359 1362 Systemic Endocrine Instigation of Indolent Tumor Growth Requires Osteopontin McAllister SS Gifford AM Greiner AL Kelleher SP Saelzer MP et al Cell 2008 133 994 1005 Chemokine Signaling via the CXCR2 Receptor Reinforces Senescence Acosta JC O Loghlen A Banito A Guijarro MV Augert A et al Cell 2008 133 1006 1018 Apocrine Cysts of the Breast Biomarkers Origin Enlargement and Relation with Cancer Phenotype Celis JE Gromov P Moreira JMA Cabezon T et al Mol Cell Proteomics 2006 5 432 483 Towards discovery driven translational research in breast cancer Celis JE Moreira JMA Gromova I Cabezon T et al FEBS J 2005 272 Cytokine expression in pediatric subperiosteal orbital abscesses Fu SY Su GY McKinley SH Yen MT Can J Ophthalmol 2007 42 865 869 Production of Chemokines by Perivascular Adipose Tissue A Role in the Pathogenesis of Atherosclerosis Henrichot E Juge Aubry CE Pernin A Pache J C et al Aterioscler Thromb Vasc Biol 2005 25 2594 2599 Viral Interactions in Human Lymphoid TissueL Human Herpesvirus 7 Suppresses the replication of CCF5 Tropic Human Immunodeficiency Virus Type 1 via CD4 Modulation Lisco A Grivel J C Biancotto A Vanpouille C et al J Virol 2007 81 2 708 717 RayBio Human Cytokine Antibody Array C Series 1000 18 20 21 22 28 24 25 26 Adipokines oversecreted by omental adipo
3. 2004 Vol 101 No 18 RayBio Human Cytokine Antibody Array C Series 1000 3 3 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 Bone Marrow Stroma Influences Transforming Growth Factor B Production in Breast Cancer Cells to Regulate c myc Activation of the Preprotachykinin I Gene in Breast Cancer Cells Oh HS Moharita A Rameshwar P Cancer Res 64 6327 6336 5 Recombinant Herpes Simplex Virus Type 1 HSV 1 Codelivering Interleukin 12p35 as a Molecular Adjuvant Enhances the Protective Immune Response against Ocular HSV 1 Challenge J Virol Mar 2005 Vol 79 No 6 6 Dysregulated Inflammatory Response to Candida albicans in a C5 Deficient Mouse Strain Alaka Mullick Miria Elias Serge Picard Philippe Gros Infect Immunity Oct 2004 p 5868 5876 7 Leukotriene B4 Strongly Increases Monocyte Chemoattractant Protein 1 in Human Monocytes Li Huang Annie Zhao Frederick Wong Julia M Ayala Jisong Cui Arterioscler Thromb Vascul Biol 2004 24 1783 1788 8 Human CD1d unrestricted NKT cells release chemokines upon Fas engagement Giroux M and Fran ois Denis Yan Xu Joseph Kulkoshy Roger j Pomerantz Blood prepublished online September 2 2004 DOI 10 1182 blood 2004 04 1537 9 Monitoring the response of orthotopic bladder tumors to granulocyte macrophage colony stimulating factor therapy using the prost
4. min per wash Dilute 20X Wash Buffer II with H5O 6 From this step place array membrane 6 marked with 6 into one well and array membrane 7 with 7 into another well 7 Prepare working solution for biotin conjugated antibodies Add 100 ul of 1x blocking buffer to the Biotin Conjugated Antibody 6 tube Mix gently and transfer all mixture to a tube containing 2 ml of 1x blocking buffer Add 100 ul of 1x blocking buffer to the Biotin Conjugated Antibody 7 tube Mix gently and transfer all mixture to a tube containing 2 ml of 1x blocking buffer Note the diluted biotin conjugated antibodies can be stored at 4 C for 2 3 days 8 Add 1 ml of diluted biotin conjugated antibodies to each membrane 1 ml of diluted biotin conjugated antibodies 6 to array membrane 6 marked with 6 and 1 ml of diluted biotin conjugated antibodies 7 to array membrane 7 marked with 7 Incubate at room temperature for 1 2 hours Note incubation may be done at 4 C for overnight 9 Wash as directed in steps 4 and 5 RayBio Human Cytokine Antibody Array C Series 1000 9 10 Add 2 ml of 1 000 fold diluted HRP conjugated streptavidin e g add 2 ul of HRP conjugated streptavidin to 1998 ul 1X Blocking Buffer to each membrane Note mix the tube containing 1 000X HRP Conjugated Streptavidin well before use since precipitation may form during storage 11 Incubate at room temperature for 2 hours Note incubation may be done at 4 C for
5. overnight 12 Wash as directed in steps 4 and 5 B Detection Do not let the membrane dry out during detection The detection process must be completed within 40 minutes without stopping 1 Proceed with the detection reaction Add 250 ul of 1X Detection Buffer C and 250 ul of 1X Detection Buffer D for one membrane mix both solutions Drain off excess wash buffer by holding the membrane vertically with forceps Place membrane protein side up 6 or 7 mark is on the protein side top left corner on a clean plastic sheet provided in the kit Transfer the mixed Detection Buffer onto the membrane and incubate at room temperature for 2 minutes Ensure that the detection mixture is completely and evenly covers the membrane without any air bubbles 2 Drain off any excess detection reagent by holding the membrane vertically with forceps and touching the edge against a tissue Gently place the membrane protein side up on a piece of plastic sheet 6 or 7 mark is on the protein side top left corner Cover with another piece of plastic sheet on the array Gently smooth out any air bubbles Avoid using pressure on the membrane RayBio Human Cytokine Antibody Array C Series 1000 10 3 Expose the array to x ray film we recommend to use Kodak X Omat AR film and detect the signal using film developer or the signal can be detected directly from the membrane using a chemiluminescence imaging system Expose the membranes fo
6. to simultaneously detect multiple cytokine expression levels from conditioned media patient s sera cell lysate tissue lysates and other sources Traditionally cytokines are detected by using ELISA however RayBiotech s approach has several advantages over ELISA First and most importantly is that our approach can detect many cytokines simultaneously Secondly sensitivity is greatly increased As little as 4 pg ml of MCP 1 can be detected using the protein array format In contrast at least 40 pg ml of MCP 1 is required to produce a clear signal in an ELISA assay Furthermore the detection range is much greater than ELISA For example the detection range of IL 2 varies from 25 to 250 000 pg ml using RayBiotech technology whereas the detection range varies only within 100 1000 fold in a typical RayBio Human Cytokine Antibody Array C Series 1000 2 ELISA Therefore the detection range is greater with protein array compared with ELISA Additionally the variation is lower than ELISA as well As determined by densitometry the variation between two spots ranged from 0 to 10 in duplicated experiments In contrast variation about 20 in ELISA is much higher Finally the system is much quicker and can be much easier to adapt to high throughput techniques Pathway specific array systems allow investigators to focus on the specific problem and are becoming an increasingly powerful tool in cDNA microarray system RayBiotech s first protein
7. 7 top left corner marked with 7 into same well of the provided eight well tray 6 or 7 marked side is the antibody printed side Add 2 ml 1X Blocking Buffer and incubate at room temperature for 30 min to block membranes Add some Blocking Buffer between the two membranes Make sure there are no bubbles between membranes Decant Blocking Buffer from each container and incubate membranes with sample at room temperature for 1 to 2 hours Dilute sample using 1X Blocking Buffer if necessary Note We recommend using 1 2 ml of undiluted conditioned media or 1 2 ml of 2 fold to 5 fold diluted sera or plasma or 200 250 ug range 50 500 ug of total protein for cell lysates and tissue lysates Dilute the lysate at least 10 fold with 1X blocking buffer Add some samples between array membrane 6 and 7 Make sure there are no bubbles between membranes RayBio Human Cytokine Antibody Array C Series 1000 8 Note The amount of sample used depends on the abundance of cytokines More of the sample can be used if the signals are too weak If signals are too strong the sample can be diluted further Note Incubation may be done at 4 C for overnight 4 Decant the samples from each container and wash 3 times with 2 ml of 1X Wash Buffer I at room temperature with shaking Please allow 5 min per wash Dilute 20X Wash Buffer I with HO 5 Wash 2 times with 2 ml of 1X Wash Buffer II at room temperature with shaking Allow 5
8. MP 7 ErbB3 b NGF Erythropoietin R EPO R BTC E Selectin IGFBP 4 IL 9 NRG1 beta 1 HRG1 beta 1 mes sun pma e leri Jem jmesann jorin _____ ____ mama joen ______ fosteoprotegorn _ Fas Ligand Fas TNFRSF6 Fer RIIB C CD27 Fractalkine CD30 FSH CD40 Furin CD40 Ligand Galectin 7 17C 1 CD23 Fc epsilon RII Follistatin CEA GCP 2 CEACAM 1 CK beta 8 1 GCSF GDF 15 MIC 1 IL 1 sRI PDGF R alpha IL 1 sRII LIGHT PDGF R beta DGF AA i Lipocalin 2 NGAL PDGF AB TREM 1 10 R beta ______ L Selectin ___________ ppGF BB TROY ANTES IL 10 R alpha smo 16 E TE TE TE TE TE Te TE 5 E 5 5 a 13 v m o SH VE i VEGF R2 VEGF R3 17B VEGF C VEGF D R RayBio Human Cytokine Antibody Array C Series 1000 14 RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical community RayBio s patent pending technology allows detection of 274 cytokines chemokines and other proteins in a single experiment Our format is simple sensitive reliable and cost effective Products include Cytokine Arrays Chemokine Arrays ELISA kits Phosphotyrosine kits EIA kits Recombinant Proteins Antibodies and custom services 1 Antibody arrays 2 Cytokine antibody array Human cytokine antibody arrays Mouse
9. array system known as RayBio Human Cytokine Antibody Array is particularly useful in comparison with the human cytokine cDNA microarray system Besides the ability to detect protein expression RayBiotech s system 1s a more accurate reflection of active cytokine levels because it only detects secreted cytokines and no amplification step 1s needed Furthermore it is much simpler faster environmentally friendlier and more sensitive Simultaneous detection of multiple cytokines undoubtedly provides a powerful tool to study cytokines Cytokines play an important role in innate immunity apoptosis angiogenesis cell growth and differentiation Cytokines are involved in most disease processes including cancer and cardiac diseases The interaction between cytokines and the cellular immune system is a dynamic process The interactions of positive and negative stimuli and positive as well as negative regulatory loops are complex and often involve multiple cytokines Without doubt simultaneous detection of multiple cytokines provides a powerful tool to study cytokines LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT6 B with effects on multiple cytokines Tang X Marciano DL Leeman SE Amar S PNAS April 5 2005 vol 102 no 14 5132 5137 2 HIV 1 mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced encephalopathy Xu Y Kulkoshy J Pomerantz RJ PNAS 2004 May 4
10. ate specific antigen gene as a reporter Wu Q Esuvaranathan K Mahendran R Clin Cancer Res 2004 Oct 15 10 20 6977 84 10 Neuroglial activation and neuroinflammation in the brain of patients with autism p NA Vargas DL Nascimbene C Krishnan C Zimmerman AW Pardo CA Ann Neurology 2005 Jan 1 DOI 10 1002 ana 20315 11 Cytokine profiling of macrophages exposed to Porphyromonas gingivalis its LPS or its FimA Zhou Q Desta T Graves DT Amar S Infect Immunity IAI 2005 Feb 73 2 935 43 RayBio Human Cytokine Antibody Array C Series 1000 4 Here s how it works Array aa support d d Sample Incubation of Sample YY With arrayed antibody 1 2 hrs Cocktail of x j fq Supports Biotin Ab KX Incubation with YYY Biotinylated Ab 1 2 hrs Labeled streptavidin 5 Incubation with 2 hrs Y Labeled streptavidin s gt Detection of signals i Data analysis and graph RayBio Human Cytokine Antibody Array C Series 1000 5 II Materials Provided Upon receipt all components of the RayBio Human Cytokine Antibody Array kit should be stored at 20 C to 80 C At 20 C to 80 C the kit will retain complete activity for up to 6 months Once thawed the array membranes and 1X Blocking Buffer should be kept at 20 C and all other component should be stored at 4 C After thawing the reagents the kit must be
11. ay C Series 1000 20 Note RayBio is the trademark of RayBiotech Inc Cytokine protein arrays are RayBiotech patent pending technology 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 three months from the date of purchase when handled and stored properly In the event of any defect in quality or merchantability RayBiotech s liability to the buyer for any claim relating to products shall be limited to replacement or refund of the purchase price Kodak X Omat is the trademark of Eastman Kodak Company This product is for research use only mmu u u uu u ajajajaja mm mmm ajaja 7 2009 RayBiotech Inc RayBio Human Cytokine Antibody Array C Series 1000 21
12. cytokine antibody arrays Rat cytokine antibody arrays Pathway or disease focused antibody arrays Inflammation antibody array Angiogensis antibody array Chemokine antibody array Growth factor antibody array MMP antibody array Atherosclerosis antibody array Adipokine antibody arrays Antibody analysis tool software ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Cytokine protein arrays Quantibody arrays for quantitative measurement of cytokine and other protein concentration 10 Phosphorylation antibody arrays 11 Biotin label based antibody arrays for high density antibody arrays 12 EIA 13 Peptide BW RayBio Human Cytokine Antibody Array C Series 1000 15 RayBiotech also provides excellent custom service l Protein arrays Peptide synthesis Production of recombinant protein and antibody Peptide arrays Phosphorylation arrays 00 1 Os tA gt N Antibody arrays ELISA EIA Assay development Just simply send your samples and we will do the assay for you 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 Cytokine Antibody Array C Series 1000 16 VI Troubleshooting guide Weak signal or no 1 Taking too much time 1 The whole Detection p
13. ecommend using per membrane o 1l ml of Conditioned media undiluted or o 1l ml of 2 fold to 5 fold diluted sera or plasma or o 50 500 ug of total protein for cell lysates and tissue lysates use 200 250 of total protein for first experiment Dilute the lysate at least 10 fold with 1 X blocking buffer Note The amount of sample used depends on the abundance of cytokines More of the sample can be used if the signals are too weak If the signals are too strong the sample can be diluted further If you experience high background you may further dilute your sample B Handling Array Membranes e Always use forceps to handle membranes and grip the membranes by the edges only e Never allow the array membranes to dry during experiments RayBio Human Cytokine Antibody Array C Series 1000 7 C Incubation Completely cover the membranes with sample or buffer during incubation and cover the eight well tray with a lid to avoid drying Avoid foaming during incubation steps Perform all incubation and wash steps under gentle rotation Several incubation steps such as step 2 blocking step 3 sample incubation step 8 biotin Ab incubation or step 11 HRP streptavidin incubation may be done at 4 C for overnight Please make sure to cover the 8 well plate tightly to prevent evaporation IV Protocol A Blocking and Incubation 1 Place one array membrane 6 top left corner marked with 6 and one array membrane
14. emark of RayBiotech Inc RayBio Human Cytokine Antibody Array C Series 1000 1 I Introduction All cell functions including cell proliferation cell death and differentiation as well as maintenance of health status and development of disease are controlled by a multitude of genes and signaling pathways 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 a disparity between the relative expression levels of mRNA and their corresponding proteins Therefore analysis of the protein profile is critical Currently two dimensional polyacrylamide SDS page coupled with mass spectrometry is the mainstream approach to analyzing multiple protein expression levels gt However the requirement of sophisticated devices and the lack of quantitative measurements greatly limit their broad application Thus effective study of multiple protein expression levels has been complicated costly are time consuming until now Our RayBio Human Cytokine Antibody Array is the first commercially available protein array system By using the RayBiotech system scientists can rapidly and accurately identify the expression profiles of multiple cytokines in several hours inexpensively The RayBiotech kit provides a simple format and highly sensitive approach
15. ize the results from different membranes being compared The signals also can be detected and quantified by using a chemiluminescence imaging device The RayBio Analysis Tool is a program specifically designed for analysis of RayBio Cytokine Antibody Arrays This tool will not only assist in compiling and organizing your data but also reduces your calculations to a copy and paste Call RayBiotech Inc at 770 729 2992 for ordering information RayBio Human Cytokine Antibody Array C Series 1000 12 RayBio Human Cytokine Antibody Array 6 60 a b f g h i i k l m n Eotaxin eer eon LIGH MCP 1 MCP MCP MCP 4 PDGF BB RANTES TARC 1 TGF f 3 TNF a TNF B PDGF BB RANTES TNF a TNF B RayBio Human Cytokine Antibody Array 7 60 a b c d e f g h i i k l m n 1 POS POS NEG NEG Blank Acrp30 AgRP Angiopoietin 2 Axl bFGF b NGF BTC CCL 28 2 POS POS NEG NEG Blank Acrp30 AgRP Angiopoietin 2 bFGF b NGF BTC CCL 28 3 CK Dtk EGF R ENA 78 Fa FRSF 4 FGF 9 SF GITR Ligan TR 5 ICAM 1 ICAM 3 IGFBP 3 IGFBP 6 IGF I SR IL 1 RA ST2 IL 1 RI IL 11 IL 12 p40 IL 12 p70 IL 17 IL 2 R alpha IL 6 R IL 8 6 ICAM 1 ICAM 3 IGFBP 3 IGFBP 6 IGF I SR IL 1 R4 ST2 IL 1 RI IL 11 IL 12 p40 IL 12 p70 IL 17 IL 2 R alpha IL 6 R IL 8 9 TIMP 1 TIMP 2 Thrombopoietin TRAIL TRAIL R4 uPAR VEGF VEGF D Blank Blank Blank Blan
16. k POS 10 TECK TIMP 1 TIMP 2 Thrombopoietin TRAIL R3 TRAIL R4 uPAR VEGF VEGF D Blank Blank Blank Blank POS o IL 12 reacts with both IL 12p40 and IL 12p70 o IL 12p70 only recognizes IL 12p70 o GRO reacts with CXCLI CXCL2 and CXCL3 GRO alpha beta and gamma respectively o GRO areacts only with CXCLI VEGF reacts with VEGF 165 and VEGF 121 o TGF beta 1 reacts only with active form of TGF beta 1 O We also offer Custom Human Cytokine Antibody Arrays You can select the cytokines of interest from the following list and we will produce the customized array at an affordable price For more information please visit our website www raybiotech com RayBio Human Cytokine Antibody Array C Series 1000 13 Human Custom Antibody Array List 285 proteins eemensro owe ome tone __ __________ ________ oeno aei ocr hwsmreorn fonaa ono Jeznape snes Activin A AFP AgRP ART ALCAM Angiopoietin 1 Decorin Angiogenin DKK 1 DKK 3 Jenn _ jemureames umes hmmeame umes Angiostatin DPPIV CD26 HVEM MMP 10 TARC ANGPTL4 AR amphiregulin DR6 Dtk E Cadherin ________ B7 1 CD80 EDA A2 ICAM 3 IL 5 R alpha lus TGF beta 2 Bate2 M EGF ENA 78 Betacellulin BTC Endoglin Endostatin BLC BMP 5 Eotaxin 2 BMP 4 Eotaxin 3 EpCAM TROP1 BMP 6 ErbB2 B
17. r 40 seconds Then re expose the film according to the intensity of signals If the signals are too strong background too high reduce exposure time e g 5 30 seconds If the signals are too weak increase exposure time e g 5 20 min or overnight Or re incubate membranes overnight with 1x HRP conjugated streptavidin and redo detection in the second day 4 Save membranes in 20 C to 80 C for future references RayBio Human Cytokine Antibody Array C Series 1000 11 V Interpretation of Results The following figure shows a representative RayBio Human Cytokine Antibody Array membrane probed with patient s plasma and Membranes were exposed to Kodak X Omat film at room temperature for 1 minute The biotin conjugated IgG produces positive signals which can be used to identify the orientation and to compare the relative expression levels among the different membranes One important parameter is background To obtain the best results we suggest that several exposures be attempted We also strongly recommend using a negative control in which the sample is replaced with an appropriate mock buffer according to the array protocol particularly during your first experiment Typical results using RayBio Cytokine Antibody arrays By comparing the signal intensities relative expression levels of cytokines can be made The intensities of signals can be quantified by densitometry The positive control can be used to normal
18. rocess must be signal for Detection completed in 30 min 2 Film developer does 2 Fix film developer not work properly 3 Did not mix HRP 3 Mix tube containing 1 000X HRP streptavidin well before Conjugated Streptavidin well before use since 1 precipitation may form during storage 4 Sample is too dilute 4 Increase sample volume e g using undilute sample or using more cells e g seed 2 million cells After 1 or 2 days change complete medium with low serum medium and collect conditioned medium 2 day later Use about 1 to 2 ml of conditioned medium for experiment 5 Other 1 Reduce blocking concentration by diluting 3 Slightly increase biotin antibody concentrations 4 Expose film for overnight to detect weak signal Uneven signal 1 Bubbles formed 1 Remove bubble during incubation during incubation 2 Membranes were not 2 Completely cover membranes with solution completely covered by solution High background 1 Exposure to x ray file 1 Decrease exposure time is too long 2 Membranes were 2 Completely cover membranes with solution experiment 3 Sample is too 3 Use more diluted sample concentrated RayBio Human Cytokine Antibody Array C Series 1000 17 Selected References Using RayBiotech Products 12 13 14 15 16 17 18 19 Classification and prediction of clinical Alzheimer s diagnosis based on plasma signaling proteins Ray S Britschgi M Herbert C
19. se tissue in human obesity Maury E Ehala Aleksejev K Guiot Y Detry R Vandenhooft A Brichard SM Am J Physiol Endocrinol Metab 2007 293 E656 E665 Atrial natriuretic peptide inhibits the production of adipokines and cytokines linked to inflammation and insulin resistance in human subcutaneous adipose tissue Moro C Klimeakova E Lolmede K Berlan M et al Diabetologia 2007 50 1038 1047 Role of Human Valve Interstitial Cells in Valve Calcification and Their Response to Atorvastatin Osman L Yacoub MH Latif N Amrani M Chester AH Circulation 2006 1 14 suppl I 1547 I552 Monocyte Chemotactic Protein 1 Mediates Prostate Cancer Induced Bone Resorption Lu Y Cai Z Xiao G Keller ET et al Cancer Res 2007 67 8 3646 3653 Innate Response by Ficolin Binding in Apoptotic Placenta is Associated with the Clinical Syndrome of Preeclampsia Wang CC Yim KW Poon TCW Choi WK et al Clin Chem 2007 53 42 52 Adaptive secretion of granulocyte macrophage colony stimulating factor GM CSF mediates imatinib and nilotinib resistance in BCR ABL progenitors via JAK 1 STAT 5 pathway activation Wang Y Cai D Brendel C Barett C Erben P et al Blood 2007 109 2147 2155 Cytokine Antibody Arrays A Promising Tool to Identify Molecular Targets for Drug Discovery Huang Combinatorial Chemistry amp High Throughput Screening 2003 6 79 99 RayBio Human Cytokine Antibody Array C Series 1000 19 RayBio Human Cytokine Antibody Arr
20. used within three months and please use the kit within six months of purchase e RayBio Human Cytokine Antibody Array membranes 2 4 8 array membranes 6 and 2 4 8 array membranes 7 e Biotin Conjugated Anti Cytokines 1 2 4 tubes each tube for two membranes 1 000X HRP Conjugated Streptavidin 50 ul 1X Blocking Buffer 25 50 ml 20X Wash Buffer I 10 20 ml 20X Wash Buffer II 10 20 ml 2X Cell Lysis Buffer 10 20 ml Detection Buffer C 1 5 2 5 ml Detection Buffer D 1 5 2 5 ml Eight Well Tray 1 each Manual Additional Materials Required Small plastic boxes or containers Orbital shaker Plastic sheet protector or SaranWrap Kodak X Omat AR film REF 165 1454 and film processor or Chemiluminescence imaging system RayBio Human Cytokine Antibody Array C Series 1000 6 III Overview and General Considerations A Preparation of Samples Use serum free conditioned media if possible If serum containing media is required use an uncultured media aliquot as a negative control sample since many types of sera contain cytokines For cell lysates and tissue lysates we recommend using RayBio Cell Lysis Buffer to extract proteins from cell or tissue e g using homogenizer Dilute 2X RayBio Cell Lysis Buffer with we recommend adding proteinase inhibitors to Cell Lysis Buffer before use After extraction spin the sample down and save the supernatant for your experiment Determine protein concentration We r
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