RayBio Rat Cytokine Antibody Array
Contents
1. 11 Cytokine Antibody Arrays A Promising Tool to Identify Molecular Targets for Drug Discovery Huang Combinatorial Chemistry amp High Throughput Screening 2003 6 79 99 12 K S Rosenthal N Goel R Singavarapu D H Zimmerman Cel 1000 protects mice against HSV 1 challenge by stimulating IL 2 production Abstract published in abstract book of Interscience Conference on Antimicrobial Agents and Chemotherapy held in Chicago during Septl4 17 2003 13 Y Lin Ruochun Huang Li Pai Chen Henry Lisoukov Zhen Hai Lu Shiyong Li Cheng C Wang and R P Huang 2003 Profiling of cytokine expression by biotin labeled based protein arrays Proteomics 3 1750 1757 14 C C Wang R P Huang H Lisoukov M Sommer R Huang Y Lin and J Burke 2002 Array based multiplexed screening and quantitation of human cytokines and chemokines J Proteome Res 1 337 343 15 R Huang Y Lin C C Wang J Gano B Lin Q Shi A Boynton J Burke and R P Huang 2002 Connexin suppresses human glioblastoma cell growth by down regulation of monocyte chemotactic protein 1 as discovered using protein array technology Cancer Research 62 2806 2812 RayBio Rat Cytokine Antibody Array Protocol 15 16 R P Huang R Huang Y Fan and Y Lin 2001 A novel method for high throughput protein profiling from conditioned media and patient s sera Ana Biochem 294 1 55 62 RayBio Rat Cytokine Antibody Array Protocol 16 Note R2. IV Protocol A Blocking and Incubation 1 Place each membrane into the provided eight well tray means the antibody printed side 2 Add 2 ml 1X Blocking Buffer and incubate at room temperature for 30 min to block membranes Note incubation may be done at 4 C for overnight 3 Incubate membranes with 1ml of sample at room temperature for 1 to 2 hours Dilute sample using 1X Blocking Buffer if necessary Note We recommend using I ml of Conditioned media or I ml of original or 10 fold diluted sera or plasma or 50 500 ug of protein for cell lysates and tissue lysates Dilute the lysate at least 10 folds 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 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 H20 5 Wash 2 times with 2 ml of 1X Wash Buffer II at room temperature with shaking Allow 5 min per wash Dilute 20X Wash Buffer II with H O 6 Prepare working solution for primary antibody RayBio Rat Cytokine Antibody Array Protocol T Add 100 ul of 1X blocking buffer to the Biotin Conjugated Anti Cytokines tube Mix gently and transfer all mixture to a tube cont
3. Rat Antibody Array 2 Detect 34 cytokines in one experiment ros os wa meo fama aoin ae eana mind oe sec ais Tee ana Fraea hati ileal gonme sais pi Th IL 13 Leptin LIX L Selectin MCP 1 MIP 3alpha PDGF AA Prolactin R yS TIMP 1 Chemokine 1 TNF alpha VEGF BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK Pos For use with serum plasma condition medium urine other body fluids cell lysates and certain tissue lysates samples 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 RayBio Rat Cytokine Antibody Array Protocol 11 Human cytokine antibody arrays Mouse 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 Quantibody arrays for quantitative measurement of cytokine and other protein concentraton Phosphory
4. multiple cytokines in several hours inexpensively The RayBiotech kit provides a simple format and highly sensitive approach to simultaneously detect multiple cytokine expression levels from conditioned media patient s sera cell lysate tissue lysates and other sources The RayBio Mouse Cytokine Antibody Array C series 1000 can detect 96 mouse cytokines in single experiment RayBiotech also provides RayBio Human Cytokine Antibody Array C series 4000 which is the only product available in the market that can detect 274 human cytokines in single experiment Traditionally cytokines are detected by using ELISA enzyme linked immunsorbent assays however RayBiotech s approach has several RayBio Rat Cytokine Antibody Array Protocol 2 advantages over ELISA First and most important our approach can simultaneously detect many cytokines Secondly the sensitivity is higher With this approach most cytokines can be detected at pg ml levels As little as 10 pg ml of human IL 2 can be detected in the protein array format Furthermore the detection range is much greater than ELISA For example the detection range of human IL 2 varies from 10 to 100 000 pg ml whereas the detection range varies only within 100 1000 fold in a typical ELISA Therefore the detection range with protein arrays is greater than ELISA Additionally variability is far lower in comparison ELISA As determined by densitometry the variation between two spots ran
5. 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 normalize the results from the 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 Rat Cytokine Antibody Array Protocol 10 RayBio Rat Cytokine Antibody Array 1 Detect 19 rat cytokines in one experiment f h a b c d e g MCP 1 MIP 30 B NGF TIMP 1 VEGF BLANK BLANK BLANK BLANK BLANK BLANK BLANK BLANK e BLANK BLANK BLANK BLANK BLANK BLANK BLANK O oN O A F Q PN RayBio
6. RayBio Rat Cytokine Antibody Array Patent Pending Technology User Manual Revised 06 14 09 RayBio Rat Cytokine Antibody Array 1 Cat AAR CYT 1 RayBio Rat Cytokine Antibody Array 2 Cat AAR CYT 2 RayBio Custom Ray Cytokine Antibody Array Cat AAR CUST RayBio Rat Cytokine Antibody Array Service Cat AAR SERV Please read manual carefully before starting experiment HOO mja uja a gegen 2 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 Protocol TABLE OF CONTENTS LTO ACM Thies teacicse case douvaveetadorepeitedunsante 2 How It W OTK Siivscniense ntstciatendamiaiveedaciuasiadts 4 II Materials Provided cccccee cece ee eees 5 Additional Materials Required 4 5 III Overview and General Considerations 6 A Preparation of Samples 0000e 6 B Handling Array Membrane 6 C Incubation ris siadcecutsncnuisietdentoidkeusenn 6 IV ig 0 00 Rn ee eee renee eee ee rn en eee net 7 A Blocking and Incubation 4 7 B Detection secdidndtancd natantededuacededisameas 9 V Interpretation of Results 5 10 VI Troubleshooting Guide 000 8 13 VII Reference Lis
7. aining 2 ml of 1X blocking buffer Note the diluted biotin conjugated antibodies can be stored at 4 C for 2 3 days 7 Add 1 ml of diluted biotin conjugated antibodies to each membrane Incubate at room temperature for 1 2 hours Note incubation may be done at 4 C for overnight 8 Wash as directed in steps 4 and 5 9 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 10 Incubate at room temperature for 2 hours Note incubation may be done at 4 C for overnight 11 Wash as directed in steps 4 and 5 RayBio Rat Cytokine Antibody Array Protocol 8 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 mark is on the protein side top left corner on a clean plastic sheet provided in the kit Pipette the mixed Detection Buffer onto the membrane and incubate at room temperature for 2 minutes Ensure that the detection mixture is completely and ev
8. ayBio 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 ECL is the trademark of Amersham Pharmacia Biotech This product is for research use only 2008 RayBiotech Inc RayBio Rat Cytokine Antibody Array Protocol 17
9. enly covering 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 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 3 Expose the array to x ray film we recommend to use Kodak x omat AR film and detect signal using film developer Or the signal can be detected directly from the membrane using a chemiluminescence imaging system Expose the membranes for 40 seconds and 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 reference RayBio Rat Cytokine Antibody Array Protocol 9 V Interpretation of Results The following figure shows RayBio Rat Cytokine Antibody Array membranes probed with conditioned media from two different cell lines Membranes were exposed to Kodak x omat film at room temperature for 1 minute The biotin conjugated IgG produces positive signals which
10. ged from 0 to 10 in duplicated experiments In contrast variation about 20 in ELISA is much higher Finally the system is much quicker and 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 systems RayBiotech s first protein array system known as RayBio Rat Cytokine Antibody Array is particularly useful in comparison 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 Cytokines play an important role in innate immunity apoptosis angiogenesis cell growth and differentiation They 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 RayBio Rat Cytokine Antibody Array Protocol 3 Here s how it works Array support i YYY E r Samples t Incubation of Sample t with arrayed antibody Cocktail of K I lt l
11. lation antibody arrays Biotin label based antibody arrays for high density antibody arrays Antibody analysis tool software ELISA Cell based phosphorylation assay Custom antibody arrays Antibody Recombinant protein Protein arrays TON ae ee RayBiotech also provides excellent custom service 1 Antibody arrays Protein arrays Peptide synthesis Production of recombinant protein and antibody Peptide arrays Phosphorylation arrays ELISA PUO acl 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 Rat Cytokine Antibody Array Protocol 12 VI Troubleshooting guide Weak signal or no 1 Taking too much time 1 The whole Detection process 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 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 experi
12. ment 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 allowed to dry out during during experiment experiment 3 Sample is too 3 Use more diluted sample concentrated RayBio Rat Cytokine Antibody Array Protocol 13 Reference List iE 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 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 Cytokine responses elicited in endothelial cells after treatment with a specific toxin Jaya Pandey BioCompare Product Review May 13 2004 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 J
13. os M A Moreira Noona Ambartsumian Kerstin Sandelin Fritz Rank and Irina Gromova Molecular Cellular Proteomics April 2004 11 3 328 39 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 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 Reduced T cell and dendritic cell function is related to cyclooxygenase 2 overexpression and protaglandin e 2 secretion in patients with breast cancer Pockaj BA Basu GD Annals of Surgical Oncology 3 327 344 2004 Inhibition of macrophage migration inhibitory factor decreases proliferation and cytokine expression in bladder cancer cells Katherine RayBio Rat Cytokine Antibody Array Protocol 14 L Meyer Siegler BMC Cancer 2004 4 34 9 The malaria metabolite hemozoin initiates proinflammatory signaling via a MyD88 dependent pathway International Congress of Immunology 2004 July W23 81 10 In Vivo Proteomic Analysis of Cytokine Expression in Laser Capture Microdissected Urothelial Cells of Obstructed Ureteropelvic Junction Procured by Laparoscopic Dismembered Pyeloplasty Journal of Endourology 2003 June Volume 17 Number 5 Page 333 336
14. rations A Preparation of Samples Use serum free conditioned media if possible If serum containing conditioned media is required use the serum as a control since many types of sera contain cytokines For cell lysates and tissue lysates we recommend using 1X Cell Lysis Buffer to extract proteins from cell or tissue e g using homogenizer After extraction spin the sample down and save the supernatant for your experiment Determine the protein concentration Dilute 2X Cell Lysis Buffer with H2O we recommend adding proteinase inhibitors to Cell Lysis Buffer before use We recommend using 1 ml of Conditioned media or 1 ml of original or 10 fold diluted sera or 50 500 ug of protein for cell lysates and tissue lysates If you experience high background you may further dilute your sample B Handling Array Membranes Always use forceps to handle membranes and grip the membranes by the edges only Never allow the array membranes to dry during experiments 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 7 biotin Ab incubation or step 10 HRP streptavidin incubation may be done at 4 C for overnight RayBio Rat Cytokine Antibody Array Protocol 6
15. t 22 45 chaecseidesceseesensieincaded 14 Cytokine protein arrays are RayBiotech patent pending technology RayBio is the trademark of RayBiotech Inc RayBio Rat Cytokine Antibody Array Protocol 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 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 Rat Cytokine Antibody Array is the first commercially available cytokine protein array system By using the RayBiotech system scientists can rapidly and accurately identify the expression profiles of
16. t 4 J Biotin Ab KK Incubation with YYY Biotinylated Ab ee Labeled eecee 4 streptavidin i Se Incubation with Y Y labeled Streptavidin 1 hrs eT Detection of signals Data analysis and graph RayBio Rat Cytokine Antibody Array Protocol 4 II Materials Provided Upon receipt all components of the RayBio Rat 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 components should be stored at 4 C After thawing the reagents the kit must be used within three months and please use the kit within six months of purchase RayBio Rat Cytokine Antibody Array membranes 2 4 8 membranes Biotin Conjugated Anti Cytokines 1 2 4 vials each vial is for two array membranes 1 000X HRP Conjugated Streptavidin 50 ul 1X Blocking Buffer 15 25ml 20X Wash Buffer I 10 20ml 20X Wash Buffer IT 10 20ml 2X Cell Lysis Buffer 10 20ml Detection Buffer C 1 5 2 5ml Detection Buffer D 1 5 2 5ml Eight Well Tray 1 each Manual Additional Materials Required or Small plastic boxes or containers Orbital shaker Plastic sheet protector or SaranWrap Kodak x omat AR film REF 165 1454 and film processor Chemiluminescence imaging system RayBio Rat Cytokine Antibody Array Protocol 5 HI Overview and General Conside
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