RayBio - BioCat
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1. cc eee eens 11 VI Troubleshooting Guide ccc cece eee 15 VII Reference Wis hacae wen inncarderonsaweeusueh veda 16 Cytokine Antibody Arrays are RayBiotech patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Mouse Cytokine Antibody Array G series 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 many genes and signaling pathways New techniques such as cDNA microarrays have enabled us to analyze the global gene expression However almost all cell functions are executed by proteins which cannot be studied by DNA and RNA alone Experimental analysis clearly shows a disparity between the relative expression levels of mRNA and their corresponding proteins Therefore it is critical to analyze the protein profile Currently two dimensional polyacrylamide SDS page coupled with mass spectrometry is the mainstream approach to analyzing multiple protein expression levels However the requirement of sophisticated devices and the lack of quantitative measurements greatly limit its broad application Thus no simple cost effective and rapid method of analysis of multiple protein expression levels has been available to researchers until now Our RayBio Rat Cytokine Antibody Array is the first commercially available rat cytokine antibody array system2. By using the RayBiotech system scientists can rapidly and accurately identify the expression profiles of multiple cytokines in several hours inexpensively The RayBiotech kit G series is a glass chip format The kit provides a highly sensitive approach to simultaneously detect multiple cytokine expression levels from cell culture supernatant patient s serum tissue lysate and other sources The arrays are manufactured using non contact arrayer The experimental procedure is simple and can be performed in any laboratory The signals from G series arrays are detected using a laser scanner Besides the products listed in this manual RayBiotech also provides RayBio Mouse Cytokine Antibody Array G series 1000 for simultaneous detection of 96 mouse cytokines in single experiment RayBiotech also provides RayBio Human Cytokine Antibody Array G series 2000 which is the only product available in the market that can detect 174 human cytokines in single experiment We also provide quantitative antibody arrays which can RayBio Mouse Cytokine Antibody Array G series 2 quantitatively measure multiple protein expression in antibody array technology Pathway specific array systems allow investigators to focus on the specific problem and are becoming an increasingly powerful tool in cDNA microarray system RayBiotech s first protein array system known as RayBio Rat Cytokine Antibody Array is particularly useful compared with the Rat cyt
3. Mahendran R Clinical Cancer Research 2004 Oct 15 10 20 6977 84 10 Neuroglial activation and neuroinflammation in the brain of patients with autism p NA Diana L Vargas Caterina Nascimbene Chitra Krishnan Andrew W Zimmerman Carlos A Pardo Annals of Neurology 2005 Jan 1 DOI 10 1002 ana 20315 11 Cytokine profiling of macrophages exposed to Porphyromonas gingivalis its LPS or its FimA Qingde Zhou Tesfahun Desta Dana T Graves and Salomon Amar Infection and Immunity IAI 2005 Feb 73 2 935 43 12 Veto like activity of mesenchymal stem cells functional discrimination between cellular responses to alloantigens and recall antigens Rameshwar P Journal of Immunology 2003 Oct 1 171 7 3426 34 13 Cytokine responses elicited in endothelial cells after treatment with a specific toxin Jaya Pandey BioCompare Product Review May 13 2004 14 Proteomic Characterization of the Interstitial Fluid Perfusing the Breast Tumor Microenvironment A Novel Resource for Biomarker and Therapeutic Target Discovery Julio E Celis Pavel Gromov Teresa Cabez n Jos M A Moreira Noona Ambartsumian Kerstin Sandelin Fritz Rank and Irina Gromova Molecular Cellular Proteomics April 2004 11 3 328 39 15 Increased Expression and Secretion of Interleukin 6 in Patients with Barrett s Esophagus Katerina Dvorakova Harinder Garewal Clinical Cancer Research 2004 Mar 15 10 6 2020 8 16 Antibody array generated profiles of cytoki
4. 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 Hyun S Oh Anabella Moharita Pranela Rameshwar CANCER RESEARCH 64 6327 6336 September 1 2004 5 Recombinant Herpes Simplex Virus Type 1 HSV 1 Codelivering Interleukin 12p35 as a Molecular Adjuvant Enhances the Protective Immune Response against Ocular HSV 1 Challenge JOURNAL OF VIROLOGY Mar 2005 Vol 79 No 6 6 Dysregulated Inflammatory Response to Candida albicans in a C5 Deficient Mouse Strain Alaka Mullick Miria Elias Serge Picard Philippe Gros Infection and Immunity Oct 2004 p 5868 5876 DOI 10 1128 TAI 72 10 5868 5876 2004 7 Leukotriene B4 Strongly Increases Monocyte Chemoattractant Protein 1 in Human Monocytes Li Huang Annie Zhao Frederick Wong Julia M Ayala Jisong Cui Arteriosclerosis Thrombosis and Vascular Biology 2004 24 1783 1788 8 Human CD1d unrestricted NKT cells release chemokines upon Fas engagement Martin Giroux and Francois Denis Yan Xu Joseph Kulkoshy Roger j Pomerantz Blood prepublished online September 2 2004 DOI 10 1182 blood 2004 04 1537 RayBio Mouse Cytokine Antibody Array G series 16 9 Monitoring the response of orthotopic bladder tumors to granulocyte macrophage colony stimulating factor therapy using the prostate specific antigen gene as a reporter Wu Q Esuvaranathan K
5. 00 ul of dye and let stand for couple hours If it is not leaking assemble 1s successful 3 Add 100 ul 1 X Blocking Buffer into each well and incubate at room temperature for 30 min to block slides Dilute 2X Blocking Buffer with H O Make sure no bubbles are in the well Note only add reagents to wells printed with antibodies 4 Decant Blocking Buffer from each well Add 50 to 100 ul of each sample to each array Incubate arrays with sample at room temperature for 1 to 2 hours Dilute sample using 1X Blocking Buffer if necessary Note We recommend using 50 to 100 ul of conditioned media or 50 to 100 wl of original or 2 5 fold diluted serum or plasma or 10 200 ug of protein for cell lysates and tissue lysates Dilute the lysate at least 10 folds with 1 X blocking buffer to make a total volume of 50 to 100 ul Make sure there is no bubble in the wells RayBio Mouse Cytokine Antibody Array G series 8 Note The amount of sample used depends on the abundance of cytokines More samples can be used if 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 5 Decant the samples from each well and wash 5 times with 150 ul of 1X Wash Buffer I at room temperature with gentle shaking 2 min per wash Dilute 20X Wash Buffer I with HO Completely remove wash buffer I in each wash step Note avoid solution flowing into neighboring wells 6 Put the glass chi
6. Bio Rat Cytokine Antibody Array kit should be stored at 20 C At 20 C the kit will retain complete activity for up to 6 months Once thawed the glass chips Fluorescent dye streptavidin and 2X Blocking Buffer should be kept at 20 C and all other component should be stored at 4 C Use within three months after reagents have been thawed Please use within six months of purchase e RayBio Rat Cytokine Antibody Microarray slides 4 subarrays e Biotin Conjugated Anti Cytokines 1 tube each tube for 4 well 1 500X Fluorescent dye Conjugated Streptavidin cy3 equivalent 1 tube 2X Blocking Buffer Sml 20X Wash Buffer I 30ml 20X Wash Buffer II 30ml 2X Cell Lysis Buffer 10 ml RayBio G series antibody array accessory including slide incubation chamber Gasket Protective cover Snap on sides and adhesive film e Manual Additional Materials Required e Orbital shaker e Laser scanner for fluorescence detection e Aluminum foil e Distilled water e Plastic box E Amiboay ML y a a Blank t gt _ Layout of Rat G series oOo O O Barcode I j 1Ij 4 arrays in one glass chip RayBio Mouse Cytokine Antibody Array G series 5 HI Overview and General Considerations A Preparation of Samples Use serum free conditioned media if possible If serum containing conditioned media is required use serum as control since many types of sera contain cytokines For cell lysates and tissu
7. RayBio Rat Cytokine Antibody Array G Series Patent Pending Technology User Manual Revised Aug 25 2008 RayBio Rat Cytokine Antibody Array G Series 2 Cat AAR CYT G1 4 RayBio Rat Cytokine Antibody Array G Series 2 Cat AAR CYT G2 4 RayBio Custom Rat Cytokine Antibody Array G Series 1 Cat AAR CUST G RayBio Rat Cytokine Antibody Array G Series 1 Service Cat AAR SER G Please read the manual carefully before you start your experiment RayBiotech Inc We Provide You With Excellent Protein Array Systems And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com RayBiotech Inc RayBio Rat Cytokine Antibody Array G Series Protocol TABLE OF CONTENTS I TO GW COM resort cra teraerebeeeniaveadseeisesiasaces 2 How It WorkS c cece cece ee ecee eee ee eee ees 4 II Materials Provided cc ccc ece cece cece nes 5 Additional Materials Required 4 5 III Overview and General Considerations 6 A Preparation of Samples 0ceee eee 6 B Handling Glass Chips 0ceee ees 6 C MCUD ANIONS asc sceanens vestoareeeseasstesetenewenses 7 IV POU CO seccetctpe ces ane tate nee tater dnenanemaneaaenes 7 A Blocking and Incubation 0068 7 B IDGLCCHON ir23psitsesatecseuluttsacntniationcerisae s 10 V Interpretation of Results
8. e lysates we recommend using 1X Cell Lysis Buffer to extract proteins from cell or tissue e g using homogenizer After extraction spin the sample and save supernatant for experiment Determine protein concentration Dilute 2X Cell Lysis Buffer with HO we recommend adding proteinase inhibitors to Cell Lysis Buffer before use Prepare relative concentrated lysate since we recommend diluting lysate at least 10 folds with Blocking Buffer for array assay We recommend using 50 to 100 ul of cell culture supernatant or 50 to 100 ul of original or 5 fold diluted serum or plasma or 20 200 ug of protein for cell lysates and tissue lysates If you experience high background you may further dilute your sample B Handling glass chips The microarray slides are sensitive do not touch the surface Hold the slides by the edges only Handle all buffers and slides with latex free gloves Avoid breaking glass slide Handle glass chip in clean environment C Incubation Completely cover array area with sample or buffer during incubation and cover the incubation chamber with adhesive film or plastic sheet protector to avoid drying Avoid foaming during incubation steps RayBio Mouse Cytokine Antibody Array G series 6 Perform all incubation and wash steps under gentle rotation Cover the incubation chamber with adhesive film during incubation particularly when incubation is more than 2 hours or 50 ul of sample or reagent is used A
9. er power and PMT parameters Inadequate reagent volumes Check pipetters and or improper dilution ensure correct preparation f ee Ensure sufficient incubation incubation step to ovemight Too low protein concentration Don t nuke too low dilution in sample Or concentrate sample Improper storage of kit Store kit at suggested temperature Excess of biotinylated antibodies Make sure correct amount of antibodies Excess of streptavidin Make sure correct amount of streptavidin Inadequate detection Check laser power And PMT parameters dust Work in clean environment Insufficient wash Increase wash time and use more wash buffer Bubbles formed dunng Avoid bubble formation incubation during incubation Arrays are not completed Completely cover arrays Covered by reagent with solution RayBio Mouse Cytokine Antibody Array G series 15 Reference List 1 LPS induces the interaction of a transcription factor LPS induced TNF a factor and STAT6 B with effects on multiple cytokines Xiaoren Tang Deborah Levy Marciano Susan E Leeman and Salomon Amar PNAS April 5 2005 vol 102 no 14 5132 5137 2 HIV 1 mediated apoptosis of neuronal cells Proximal molecular mechanisms of HIV 1 induced encephalopathy Yan Xu Joseph Kulkoshy Roger j Pomerantz PNAS 2004 May 4 2004 Vol 101 No 18 3 Synergistic increases in intracellular Ca 2 and the release of MCP 1 RANTES and IL 6 by astrocytes treated with opiates and HIV 1 Tat GLIA 2005 Apr 15
10. ne release from THP 1 leukemic monocytes exposed to different amphotericin B formulations Turtinen LW Prall DN Bremer LA Nauss RE Hartsel SC Antimicrobial Agents Chemotherapy 2004 Feb 48 2 396 403 17 The promise of cytokine antibody arrays in drug discovery process R P Huang W Yang D Yang L Flowers I R Horowitz X Cao and R Huang Expert opinion on drug discovery 2005 9 601 615 RayBio Mouse Cytokine Antibody Array G series 17 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 buyer for any claim relating to products shall be limited to replacement or refund of the purchase price RayBio Mouse Cytokine Antibody Array G series 18 This product is for research use only 2008 RayBiotech Inc RayBio Mouse Cytokine Antibody Array G series 19
11. okine cDNA microarray system Besides the ability to detect protein expression RayBiotech s system is a more accurate reflection of active cytokine levels because it only detects secreted cytokines and no amplification step is 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 RayBio Mouse Cytokine Antibody Array G series 3 Here s how it works Array Ea support axy Sample lt Incubation of Sample f j YYY With arrayed antibody 1 2 hrs S t Cocktail of i y a ae TEPOS Biotin Ab K Sy Incubation with Biotinylated Ab 1 2 hrs Labeled 4 aaaa streptavidin E Incubation with vYY Labeled streptavidin i j Y Detection of signals Data analysis and graph JI I RayBio Mouse Cytokine Antibody Array G series II Materials Provided Upon receipt all components of the Ray
12. ol Positive control is biotinylated protein It can be used to normalize the streptavidin incubation step If the positive signals from different array membranes are similar positive control is a simple and effective way for normalization Negative control Negative control is BSA Normally it should only give a background reading RayBio Mouse Cytokine Antibody Array G series 12 RayBio Rat Antibody Array G Series 1 Detect 19 cytokines in one experiment c d f g h i a b e oar wn RayBio Rat Cytokine Antibody Array G series 2 for simultaneous detection of 34 rat cytokines A B Cc D E F G H J K L i Thymus IL 13 L Selectin MCP 1 MIP 3alpha mmP 8 PDGF AA Prolactin R ma Chemokine 1 TIMP 1 Abbreviations IP 10 Interferon inducible protein 10 LAP latency associated peptide TGF 1 LIF leukocyte inhibitory factor MMP Matrix Metalloproteinase Pos positive control Neg negative control All other are used standard abbreviations 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 over 180 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 Recombinant Proteins Antibodies and custom
13. or incubate in dark room 12 Incubate at room temperature for 1 to 2 hours Note incubation may be done at 4 C for overnight 13 Wash with Wash Buffer I twice as directed in steps 5 B Fluorescence Detection 1 Decant excess Wash Buffer from wells 2 Disassemble the slide out of the incubation frame and chamber 3 Place the whole slide in a 50 ml centrifuge tube add enough Wash Buffer I about 30 ml to cover the whole slide and gently shake at room temperature for 10 minutes Decant Wash Buffer I Repeat Wash Buffer I once Wash with Wash Buffer II about 30 ml with gentle shake at room temperature for 10 minutes Or wash using slide chamber Rinse the slide with distilled H20 4 Remove water droplets by centrifuge at 1 000 rpm for 3 minutes and then let slide dry completely in air at least 20 minutes protect from light Make sure the slides are absolutely dry before the scanning procedure 5 Image the signals using laser scanner such Axon GenePix using cy3 channel Note we recommend scanning slides right after experiment You also can store the slide at 20 C in dark for several days If you do not have a laser scanner RayBioteh can provide service for you Just simply send your slide to us and we will take care of it RayBio Mouse Cytokine Antibody Array G series 10 V Interpretation of Results The following figure shows RayBio Rat Cytokine Antibody Array G 1 probed with different cell culture supernatan
14. p with frame into a box with 1X Wash Buffer I cover the whole glass slide and frame with Wash Buffer I and wash at room temperature with gentle shaking for 20 min 7 Decant the 1X Wash Buffer I from each well Put the glass chip with frame into the box with 1X Wash Buffer II cover the whole glass slide and frame with Wash Buffer II and wash 2 times at room temperature with gentle shaking for 5 min Remove all of Wash Buffer II in the well Dilute 20X Wash Buffer II with H O 8 Prepare working solution for biotin conjugated antibodies After brief spinning Add 300 ul of 1x blocking buffer to the Biotin Conjugated Antibody tube Mix gently Note the diluted biotin conjugated antibodies can be stored at 4 C for 2 3 days 9 Add 70 wl of diluted biotin conjugated antibodies to each corresponding well Incubate at room temperature for 2 hours Note incubation may be done at 4 C for overnight 10 Wash as directed in steps 5 and then wash 3 times with 150 ul of 1X Wash Buffer II at room temperature with shaking 2 min per wash Completely remove wash buffer II in each wash step RayBio Mouse Cytokine Antibody Array G series 9 11 Add 70 ul of 1 500 fold diluted Fluorescent dye conjugated streptavidin after brief spinning add 1 5 ml of Blocking Buffer to Fluorescent dye conjugated streptavidin tube to each array Cover the incubation chamber with Adhesive film Cover the plate with aluminum foil to avoid exposure to light
15. services 1 Antibody arrays Cytokine antibody array Human cytokine antibody arrays Mouse cytokine antibody arrays Rat cytokine antibody arrays Pathway or disease focused antibody arrays RayBio Mouse Cytokine Antibody Array G series 13 Inflammation antibody array Angiogensis antibody array Chemokine antibody array Growth factor antibody array MMP antibody array Atherosclerosis antibody array Quantibody arrays for quantitative measurement of cytokine and other protein concentraton Phosphorylation antibody arrays Biotin label based antibody arrays for high density antibody arrays Antibody analysis tool software Oe OS eh ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Peptide Protein arrays RayBiotech also provides excellent custom service by SF Se ae Antibody arrays Protein arrays Peptide synthesis Production of recombinant protein and antibody Peptide arrays Phosphorylation arrays ELISA Just simply send your samples and we will do the assay for you Technology transfer program Have you developed technologies or reagents interested to the scientific and research community RayBiotech can help you commercialize your technologies reagents and dream RayBio Mouse Cytokine Antibody Array G series 14 VI Troubleshooting guide Problem Weak signal Uneven signal Cause Recommendation Inadequate detection Check las
16. t The images were captured using laser scanner The biotin conjugated protein produces positive signals which can be used to identify the orientation and to compare the relative expression levels among the different wells The signal intensities obtained from laser scanner can simply be imported into our analysis tool The analysis tool will help you Locate your signal intensities to antibody array map Link the protein to website for more detailed information on the particular protein Protein list sorting Average signal intensities Subtract background Normalize the data from different samples Obtain protein level comparison charts among different samples This analysis tool is very simple and affordable which will not only assist in compiling and organizing your data but also reduces your calculations to a copy and paste step Conditioned medium Negative Control RayBio Mouse Cytokine Antibody Array G series 11 If you do not use our RayBio Analysis Tool you can locate the cytokines by referring to RayBio Rat cytokine Antibody Array G series 1 Normalization and comparison For biomarker discovery or for analysis of large number of arrays great attention must be paid to the normalization Our antibody array design includes several controls for normalization and comparison of arrays performing in different membranes and different experiments for more information please read the reference 17 Positive contr
17. void cross contamination from overflowing solution to neighboring wells Several incubation steps such as step 3 blocking step 4 sample incubation step 9 biotin Ab incubation or step 12 Fluorescent dye streptavidin incubation may be done at 4 C for overnight Please make sure to cover the incubation chamber tightly to prevent evaporation IV Protocol A Blocking and Incubation 1 Take the glass chip out from the box Let air dry for 2 hours 2 Assemble the glass chip into incubation chamber and incubation frame as shown below Note if you slide has be assembled you can go to step 3 directly Instructions for incubation chamber assembly G Series and Q series arrays Incubation chamber gt Gasket normally _ gt attached to chamber Protective Cover can Pe as be discarded EEEE Snap on sides med RayBio Mouse Cytokine Antibody Array G series 7 Carefully place slide at bottom of the chamber as shown The slide will adhere somewhat to the bottom Warning the slide is fragile so do not apply more than gentle force to the apparatus While 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 procedure with the other side You can practice assembling device using a blank glass slide After assembled add 1
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