Mouse Cytokine Antibody Array G series 1000
Contents
1. 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 Human Cytokine Antibody Array G series 1000 17 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 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 22. 12p40 and IL 12p70 IL 12p70 only recognizes IL 12p70 Abbreviations IP 10 Interferon inducible protein 10 LAP latency associated peptide TGF B1 LIF leukocyte inhibitory factor MMP Matrix Metalloproteinase Pos positive control Neg negative control All other are used standard abbreviations CRG 2 CTACK CXCL16 DPPIV Dtk _ ____ _ _ Eotaxin ___ Eotaxin 2 GITR GM CFS____ HGF R_________JICAM 1 IFN gamma __ IGFBP 2____ IGFBP 3 ___ IL 3 Rbeta MIG ________ MIP 1 alpha __ MIP 1 gamma ___ MIP2 _____ MIP 3 alpha _ MIP 3 beta _ MMP 2_____ Resistin ___ SCF________ SDF 1 alpha ____ Shh N______ sTNF RII _____ sTNFRI ____ TARC ___ _ 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 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 Inflammation antibody array Angiogensis antibody array RayBio Human Cytokine Antibody Array G series 1000 14 Chemokine antibody array Growth factor
3. 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 Cabezon 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 cytokine 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 Human Cytokine Antibody Array G series 1000 18 Note RayBio is the trademark of RayBiotec
4. 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 array III and array IV 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 ul 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 Human Cytokine Antibody Array G series 1000 8 Note The amount of sample used depends on the abundance of cytokines More sample 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 Note when transfer IC use 0 1 ul to 2 ul pipettor 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 H O Completely remove wash buffer I in each wash step Note avoid solution flowing into neighboring wells 6 Wash 2 times with 150 ul of 1X Wash Buffer II at room temperature
5. Cytokine Antibody Arrays are RayBiotech patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Human Cytokine Antibody Array G 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 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 Mouse 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 i
6. RayBio Mouse Cytokine Antibody Array G Series 1000 Patent Pending Technology User Manual Revised 07062006 RayBio Mouse Cytokine Antibody Array G series 1000 Combination of Arrays III amp IV Cat AAM CyT G1000 RayBio Mouse Cytokine Antibody Array G Series III Cat AAM CYT G3 RayBio Mouse Cytokine Antibody Array G Series IV Cat AAM CYT G4 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 Mouse Cytokine Antibody Array G Series 1000 Protocol TABLE OF CONTENTS I Introduction A EE Eke 2 How It WorkS ccc ccc cece ee ecee eee ee kk 4 II Materials Provided EC C CE EEE 5 Additional Materials Required 5 III Overview and General Considerations 6 A Preparation of Samples E jj j jU 6 B Handling Glass Chips cceee ees 6 C Incubation C L KK 7 EV Protocol s k n 445240502 5 heba d h eya d k 7 A Blocking and Incubation 7 B Detection rsi 44 cs45 sukak sieisen noraa 10 V Interpretation of Results c cece eee 11 VI Troubleshooting Guide cccc cena es 16 VIL Reference List j sik cuna lk kanal k AA dete 17
7. antibody array MMP antibody array Atherosclerosis antibody array Antibody analysis tool software ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Cytokine protein arrays NYHA MB WN RayBiotech also provides excellent custom service 1 Antibody arrays Protein arrays Peptide synthesis Production of recombinant protein and antibody Peptide arrays Phosphorylation arrays ELISA NYA ES Q N 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 Human Cytokine Antibody Array G series 1000 15 VI Troubleshooting guide Problem Weak signal Uneven signal Cause Recommendation Inadequate detection Check laser 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
8. e 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 RayBio Human Cytokine Antibody Array G series 1000 10 scanner RayBioteh can provide service for you Just simply send your slide to us and we will take care of it V Interpretation of Results The following figure shows RayBio Mouse Cytokine Antibody Array G series 1000 probed with different cell culture supernatant 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 internal control IC can als
9. es several controls for normalization and comparison of arrays performing in different membranes and different experiments for more information please read the reference 17 Positive control 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 Human Cytokine Antibody Array G series 1000 12 O NOANA RA QN o oN Oo A DY RayBio Mouse Cytokine Antibody Array G series 3 62 A B c D E F G H I J L M N RayBio Mouse Cytokine Antibody Array G series 4 34 C D E G H l J K POS 3 bFGF DPPIV CD26 E Selectin Fc gamma RIIB _ Fit 3 Ligand POS 1 POS 2 POS 3 bFGF DPPIV CD26 E Selectin Fe gamma RIIB _ Fit 3 Ligand MMP 2 MMP 3 Osteopontin Osteoporotegerin Pro MMP 9 Shh N_ Thymus CK 1 TIMP 2 TRANCE MMP 2 MMP 3 Osteopontin Osteoporotegerin Pro MMP 9 Shh N Thymus CK 1 TIMP 2 TRANCE RayBio Human Cytokine Antibody Array G series 1000 13 We also offer Custom Mouse 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 Note IL 12 reacts both IL
10. h 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 ECL is the trademark of Amersham Pharmacia Biotech RayBio Human Cytokine Antibody Array G series 1000 19 RayBio Human Cytokine Antibody Array G series 1000 20 This product is for research use only 2004 RayBiotech Inc RayBio Human Cytokine Antibody Array G series 1000 21
11. ighboring wells e Several incubation steps such as step 3 blocking step 4 sample incubation step 8 biotin Ab incubation or step 11 Alexa Flour 555 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 60 minutes 2 Assemble the glass chip into incubation chamber and incubation frame as shown below Step one align the glass chip arrays and barcode are at the same side with incubation chamber the narrow side of chamber in barcode side making sure that the array area is faced up and not covered by incubation chamber Step two loose the frame and insert the glass chip and incubation chamber into incubation frame Step three tighten the incubation frame the glass chip and incubation chamber RayBio Human Cytokine Antibody Array G series 1000 7 You can practice assembling device using a blank glass slide After assembled add 100 ul of dye and let stand for couple hours If it is not leaking assemble is successful Glass chip Incubation Frame Chamber 16 wells Wide side aTow side Wide side 4 subarrays alll Narrow side ee a Step 2 Step 1 Loose the frame and Step 3 Align glass chip insert glass chip and Tighten the frame to the chamber incubation chamber into the frame 3 Add 100 ul 1 X Blocking
12. ltiple 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 Human Cytokine Antibody Array G series 1000 3 Here s how it works Array a support f Sample lt Incubation of Sample f YYY With arrayed antibody 1 2 hrs Cocktail of W il o gt Biotin Ab KKAK Y KX Incubation with YYY Biotinylated Ab 1 2 hrs Labeled Beea 4 streptavidin E Incubation with 2 hrs YvYY Labeled streptavidin V ee gt Detection of signals U Data analysis and graph RayBio Human Cytokine Antibody Array G series 1000 4 II Materials Provided Upon receipt all components of the RayBio Mouse 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 Alexa Flour 555 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 reage
13. nexpensively 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 The RayBio Mouse Cytokine Antibody Array G series 1000 can detect 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 RayBio Human Cytokine Antibody Array G series 1000 2 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 Mouse Cytokine Antibody Array is particularly useful compared with the Mouse cytokine 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 mu
14. nts have been thawed Please use within six months of purchase e RayBio Mouse Cytokine Antibody Microarray slides 4 8 subarray III and 4 8 subarray IV e Biotin Conjugated Anti Cytokines 1 2 tubes each tube for 4 wells 1 500X Alexa Flour 555 Conjugated Streptavidin 1 2 tubes each tube for 4 wells 2X Blocking Buffer 4 ml 8ml 20X Wash Buffer I 30m1 60ml 20X Wash Buffer II 20m1 40ml 2X Cell Lysis Buffer 10 ml 16 ml Manual Additional Materials Required Accessory RayBio G series antibody array accessory including slide incubation chamber slide frame and adhesive film Cat G CFF Slide incubation chamber Cat G CHA Slide frame Cat G FRA Adhesive film Cat G FIL Orbital shaker Laser scanner for fluorescence detection Aluminum foil Distilled water Plastic box RayBio Human Cytokine Antibody Array G series 1000 5 Layout of Mouse G series 1000 Bo L Array VI r r E g Array VII mn C Blank _ _ Blank _ _ Oo O Oo O Di Barcode j jj Barcode ITIL LI 4 arrays in one glass chip III Overview and General Considerations A Preparation of Samples e Use serum free conditioned media if possible e Ifserum containing conditioned media is required use serum as control since many types of sera contain cytokines e For cell lysates and tissue lysates we recommend using 1X Cell Lysis Buffer to extract proteins fr
15. o be used to normalize the signal intensities among array membranes in different experiments 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 e 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 Mouse cytokine antibody array G series 1000 Motuse cell culture supernatant Mouse cytokine antibody array III Mouse cytokine antibody array IV Negative control RayBio Human Cytokine Antibody Array G series 1000 11 If you do not use our RayBio Analysis Tool you can locate the cytokines by referring to RayBio Mouse cytokine Antibody Array G series 1000 Please keep in mind that G series 1000 consists of two individual arrays Mouse cytokine antibody array VI and Mouse cytokine antibody array VII Refer to corresponding map 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 includ
16. om 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 e 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 e The microarray slides are sensitive do not touch the surface Hold the slides by the edges only e Handle all buffers and slides with latex free gloves RayBio Human Cytokine Antibody Array G series 1000 6 e Avoid breaking glass slide e Handle glass chip in clean environment C Incubation e 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 e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle rotation e 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 e Avoid cross contamination from overflowing solution to ne
17. 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 Human Cytokine Antibody Array G series 1000 16 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 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
18. with shaking 2 min per wash Dilute 20X Wash Buffer II with HO Completely remove wash buffer II in each wash step 7 Prepare working solution for biotin conjugated antibodies After brief spinning Add 300 ul of 1x blocking buffer to the Biotin Conjugated Antibody III tube Mix gently Add 300 ul of 1x blocking buffer to the Biotin Conjugated Antibody IV tube Mix gently Note the diluted biotin conjugated antibodies can be stored at 4 C for 2 3 days 8 Add 70 wl of diluted biotin conjugated antibodies to each corresponding well 70 ul of diluted biotin conjugated antibodies III to each subarray II 70 ul of diluted biotin conjugated antibodies IV to each subarray IV Incubate at room temperature for 2 hours Note incubation may be done at 4 C for overnight RayBio Human Cytokine Antibody Array G series 1000 9 9 Wash as directed in steps 5 and 6 10 Add 70 ul of 1 500 fold diluted Alexa Flour 555 conjugated streptavidin after brief spinning add 1 5 ml of Blocking Buffer to Alexa Flour 555 conjugated streptavidin tube to each subarray Cover the incubation chamber with Adhesive film Cover the plate with aluminum foil to avoid exposure to light or incubate in dark room 11 Incubate at room temperature for 1 to 2 hours Note incubation may be done at 4 C for overnight 12 Wash with Wash Buffer I twice as directed in steps 5 B Fluorescence Detection 1 Decant excess Wash Buffer from wells 2 Disassembl
Download Pdf Manuals
Related Search