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1. Note incubation may be done at 4 C for overnight 12 Wash with Wash Buffer 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 50 ml centrifuge tube add enough Wash Buffer about 30 ml to cover the whole slide and gently shake at room temperature for 10 minutes Decant Wash Buffer Repeat Wash Buffer once Wash with Wash Buffer Il 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 RayBio Human Apoptosis Antibody Array G series 11 y pop 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 we 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 Human Apoptosis Antibody Array using cell lysates from induced and uninduced Jurkat cell line The images were captured using laser scanner The biotin con2. 20 C At 20 C the kit will retain full activity for up to 6 months Once thawed the glass chips Alexa Flour 555 streptavidin Internal Control Proteinase Inhibitor 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 after purchase 1 RayBio Human Apoptosis Antibody Microarray slides 4 subarray wells in one slide 2 Biotin Conjugated Anti Apoptosis Antibody cocktail mix 1 vial for 4 wells of each slide 1 500X Alexa Flour 555 Conjugated Streptavidin 1 vial 2X Blocking Buffer 10 ml 20X Wash Buffer 30ml 20X Wash Buffer I 30ml 2X Cell Lysis Buffer 10 ml Proteinase Inhibitor 1 vial RayBio G series antibody array accessory including slide incubation chamber Gasket Protective cover Snap on sides and adhesive film 10 Manual ab ve e Ue id Additional Materials Required e Orbital shaker e Laser scanner for fluorescence detection e Aluminum foil e Distilled water e Plastic box RayBio Human Apoptosis Antibody Array G series 6 Layout of RayBio Human Apoptosis Antibody Array Blank gt Blank NUE SEE ETE mie NN OOOOOO Barcode LIII Il Barcode IMMM 4 arrays in one glass chip Ill Overview and General Considerations A Preparation of Samples e For cell lysates and tissue lysates we reco
3. RayBio Human Apoptosis Antibody Array G Series User Manual RayBio Human Apoptosis Antibody Array Cat AAH APO G1 4 Cat AAH APO G1 8 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 Human Apoptosis Array G series Protocol TABLE OF CONTENTS PEE Inilieell eio n How It WOIKS ais ice rt net e tne o Rao Exs Lus I Materials Provided cccceceeseeeeeeeeeeeeees Additional Materials Required Ill Overview and General Considerations A Preparation of Samples B Handling Glass Chips Ce IMCUD ANON MET We PFOCCO RR RU A Blocking and Incubation B DGC CON V Interpretation of Results ss VI RayBio Analysis Tool MIL Anay IMIabssesseususexdpentsabaSu ei au nu Ren xn Een VIII Troubleshooting Guide IX Reference List essere RayBio is the trademark of RayBiotech Inc RayBio Human Apoptosis Antibody Array G series 1 y pop I Introduction Apoptosis is the process of programmed cell death that involves a series of biochemical events leading to a characteristic
4. cell morphology and death including blebbing and changes to the cell membrane such as loss of membrane asymmetry and attachment cell shrinkage nuclear fragmentation chromatin condensation and chromosomal DNA fragmentation Apoptotic studies have increased substantially since the early 1990s In addition to its importance as a biological phenomenon such as cell termination homeostasis development and lymphocyte interactions deregulation of apoptosis has been implicated in many diseases Excessive apoptosis causes hypotrophy such as in ischemic damage whereas an insufficient apoptosis results in uncontrolled cell proliferation such as HIV progression and cancer development Apoptosis is mediated by a diverse range of cell signals both extracellular and intracellular Extracellular signals may include toxins hormones growth factors nitric oxide or cytokines Intracellular apoptotic signaling may be induced in response to stress via heat radiation nutrient deprivation viral infection hypoxia and increased intracellular calcium concentration or the binding of nuclear receptors by glucocorticoids These signals may positively or negatively induce apoptosis Two apoptotic signal transduction pathways in mammals have been reported the TNF induced model and the Fas Fas ligand mediated model TNF is the major extrinsic mediator of apoptosis Most cells in the human body have two receptors for TNF TNF H1 and TNF R2 The binding of TNF
5. dria Genes amp Dev 21 3095 3109 4 Thompson CB 1995 Apoptosis in the pathogenesis and treatment of disease Science 267 5203 1456 62 5 Br ne B 2003 Nitric oxide NO apoptosis or turning it ON Cell Death Differ 10 8 864 9 6 Fesik SW Shi Y 2001 Controlling the caspases Science 294 5546 1477 8 7 Dejean LM Martinez Caballero S Manon S and Kinnally KW 2006 Regulation of the mitochondrial apoptosis induced channel MAC by BCL 2 family proteins Biochim Biophys Acta 1762 2 191 201 8 Wajant H 2002 The Fas signaling pathway more than a paradigm Science 296 5573 1635 6 9 Chen G and Goeddel DV 2002 TNF R1 signaling a beautiful pathway Science 296 5573 1634 5 10 Murphy KM Ranganathan V Farnsworth ML Kavallaris M and Lock RB 2000 Bcl 2 inhibits Bax translocation from cytosol to mitochondria during drug induced apoptosis of human tumor cells Cell Death Differ 7 1 102 11 11 Susin SA Lorenzo HK and Zamzami N 1999 Molecular characterization of mitochondrial apoptosis inducing factor Nature 397 6718 441 6 12 Nagata S 2000 Apoptotic DNA fragmentation Exp Cell Res 256 1 12 8 RayBio Human Apoptosis Antibody Array G series 17 Note RayBio is the trademark of RayBiotech Inc Apoptosis antibody arrays are RayBiotech patent pending technology This product is intended for research only and is not to be used for clinical diagnosis Our pro
6. duces 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 Apoptosis Antibody Array G series 18 y pop This product is for research use only 2004 RayBiotech Inc RayBio Human Apoptosis Antibody Array G series 19
7. e when transfer IC please 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 at room temperature with gentle shaking 2 min per wash Dilute 20X Wash Buffer with H2O Completely remove Wash Buffer in each wash step Note avoid solution flowing into neighboring wells 6 Wash 2 times with 150 ul of 1X Wash Buffer Il at room temperature with gentle shaking 2 min per wash Dilute 20X Wash Buffer with H2O 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 mix vial Mix gently Note the diluted biotin conjugated antibodies can be stored at 4 C for 2 3 days RayBio Human Apoptosis Antibody Array G series 10 8 Add 70 ul of diluted biotin conjugated antibodies to each corresponding well Incubate at room temperature for 2 hours gt Note incubation may be done at 4 C for overnight 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 gt
8. eins IAPs and deactivates them preventing the IAPs from arresting the apoptotic process and therefore allowing apoptosis to proceed IAP also normally suppresses the activity of a group of caspases which carry out the degradation of the cell therefore the actual degradation enzymes can be seen to be indirectly regulated by mitochondrial permeability Cytochrome c is also released from mitochondria due to formation of a channel MAC in the outer mitochondrial membrane and serves a regulatory function as it precedes morphological change associated with apoptosis Once cytochrome c is released it binds with Apaf 1 and ATP which then bind to pro caspase 9 to create a protein complex Known as an apoptosome The apoptosome cleaves the pro caspase to its active form of caspase 9 which in turn activates the effector caspase 3 The tumor suppressor protein p53 also plays critical role in apoptosis p53 accumulates in response to DNA damage via interferon alpha and interferon beta pathways which induce transcription of the p53 gene and result in the increase of p53 protein level and enhancement of cancer cell apoptosis p53 prevents the cell from replicating by stopping the cell cycle at G1 or interphase to give the cell time to RayBio Human Apoptosis Antibody Array G series 3 repair however it will induce apoptosis if damage is extensive and repair efforts fail Any disruption to the regulation of the p53 or interferon genes will result in i
9. ion Negative control Negative control is BSA Normally it should only give a background reading RayBio Human Apoptosis Antibody Array G series 14 V Array Map RayBio Human Apoptosis Antibody Array G series 1 8 8 9 E F8 8 01 3 K 1 M 1 Pei Pond f res we Neg ts bax sez botw 81D em Caspases caspase 2 Pei Pos 3 Neg Neg tad e bez bow BID em Caspases caspases ope op caez cec Dae Fas Fas neg Hsezr Wseeo nsero mma iera 4 ce cow caez cec Dae Fas Fas neg Heper Wseeo sero mma ieri s orn iore eres icr ea iara icrses crepe ig wn sm sm psa Sw 6 Gri ereen terbez rores icrep GFP s tras rrt in p1 oer oss Smac 7 Survivin smwemr sme TNFalpha TNF beta TRALA TRALR2 TRALRS TRALA Xue Nu s M9 e Suvin suem sez TNFalpha Tute TRALRA TRALR2 TRALRS Tacna xar no no no We also offer sample testing service and Custom Human Apoptosis Antibody Arrays at an affordable price For more information please visit our website www raybiotech com RayBio Human Apoptosis Antibody Array G series 15 VI Troubleshooting guide Problem Cause Recommendation Check laser power Weak signal Inadequate detection Check PMT parameters Inadequate reagent volume Check pipettor Improper dilution Ens
10. jugated 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 also be used to normalize the signal intensities among array membranes in different experiments RayBio Human Apoptosis Antibody Array G series 12 Uninduced Jurkat Cell Induced Jurkat Cell Bax 2 Bcl 2 Bcl 2 Bim Caspase 3 _ lt lt a Cyto C m ag Smac Survivin Smac XIAP CIAP2 C Western Results Fig 1 Apoptotic protein profiling in Induced and uninduced Jurkat cell lines Jurkat cells were treated with apoptosis inducer set for 7 hours 10mM Actinomycin D 2mM Camptothecin 100mM Cycloheximide 10mM Dexamethasone and 100mM Etopiside 50 ug of cell lysate from both induced and uninduced Jurkat cells were incubated overnight with RayBio Human Apoptosis Antibody Array slide The antibody array slides were then washed and cocktail of biotinylated antibody mix was used to detect apoptosis related proteins After incubation with fluorescence dye the signals were scanned with fluorescence scanner Array results are shown in Figure A for uninduced Jurkat cells and Figure B for induced Jurkat cells Representative markers are highlighted in the numbered rectangular boxes Figure C shows Western Blotting of apoptotic markers Bcl 2 Caspase 3 HTRA 2 using both induced 0 Hr and induced 7 Hr Jurkat cell l
11. mmend 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 H2O 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 Protease Inhibitor Cocktail Briefly spin down the Protease Inhibitor Cocktail tube before use Add 60 ul of 1X Lysis Buffer into the vial to prepare a 100X Protease Inhibitor Cocktail Concentrate e 2X Cell Lysis Buffer Cell lysis buffer should be diluted 2 fold with deionized or distilled water before use Add 20 ul of prepared 100X Protease Inhibitor Cocktail Concentrate bring the tube to room temperature to thaw the solution before use into 1 98 ml 1X Lysis Buffer before use Mix well e We recommend using 500 to 600 ug of protein for cell lysates and tissue lysates RayBio Human Apoptosis Antibody Array G series 7 y pop The kit can also be used for serum and plasma samples as well as conditioned medium 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 glas
12. mpaired apoptosis and the possible formation of tumors A recent report has shown the involvement of IGFBPs insulin like growth factor binding protein in apoptosis IGFBP1 protein localizes to mitochondria where it binds to the BAK and hinders BAK activation and apoptosis induction When IGFBP1 is in a complex with BAK formation of a proapoptotic p53 BAK complex and apoptosis induction is impaired both in cultured cells and in liver In contrast livers of IGFBP1 deficient mice exhibit spontaneous apoptosis that is accompanied by p53 mitochondrial accumulation and BAK oligomerization These results identify IGFBP1 as a negative regulator of the BAK dependent pathway of apoptosis whose expression integrates the transcriptional and mitochondrial functions of the p53 tumor suppressor protein RayBio Human Apoptosis Antibody Array G series 4 y pop Here s how it works Array i E support aa e Samples c Incubation of Sample i YYY With arrayed antibody Overnight Supports Cocktail of Biotin Ab KAKA Y KAK Incubation with YYY Biotinylated Ab gt 2hrs Labeled 8 amp NE da streptavidin 6 Incubation with 2 hrs Y Labeled streptavidin we Detection of signals Data analysis and graph an RayBio Human Apoptosis Antibody Array G series 5 Il Materials Provided Upon receipt all components of the RayBio Human Apoptosis Antibody Array kit should be stored at
13. r 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 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 H2O Make sure no bubbles are in the well Note only add reagents to wells printed with antibodies RayBio Human Apoptosis Antibody Array G series 9 4 Decant Blocking Buffer from each well Add 50 to 100 ul of each sample to array wells Incubate arrays with sample at 4 C overnight Dilute sample using 1X Blocking Buffer if necessary Add 100 ul of Blocking Buffer to IC tube mix well and transfer 1 ul of IC to each well 50 to 100 ul of sample Note 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 Note The amount of sample used depends on the abundance of Apoptotic molecules More sample can be used if signals are too weak If signals are too strong the sample needs further dilution Note Incubation may be done for 2 hours at room temperature gt Not
14. s 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 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 Avoid cross contamination from overflowing solution to neighboring wells 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 T Take the glass chip out from the box Let air dry for 60 minutes RayBio Human Apoptosis Antibody Array G series 8 y pop 2 Assemble the glass chip into incubation chamber and incubation frame as shown below Note if you slide has been pre assembled you can go to step 3 directly Instructions for incubation chamber assembly G Series and Q series arrays Incubation chamber Gasket normally attached to chamber Protective Cover can EH T be discarded RE TESA Snap on sides SS Carefully place slide at bottom of the chambe
15. to TNF H1 has been shown to initiate the pathway that leads to caspase activation via the intermediate membrane proteins TNF receptor associated death domain TRADD and Fas associated death domain protein FADD Binding of this receptor can also indirectly lead to the activation of transcription factors involved in cell survival and inflammatory responses The Fas receptor also known as Apo 1 or CD95 binds the Fas ligand The interaction RayBio Human Apoptosis Antibody Array G series 2 y pop between Fas and FasL results in the formation of the death inducing signaling complex DISC which contains the FADD caspase 8 and caspase 10 Following TNF H1 and Fas activation in mammalian cells a balance between pro apoptotic BAX BID BAK or BAD and anti apoptotic Bcl X and Bcl 2 members of the Bcl 2 family is established This balance is the proportion of pro apoptotic homodimers that form in the outer membrane of mitochondrion The pro apoptotic homodimers are required to make the mitochondrial membrane permeable for the release of caspase activators such as cytochrome c and SMAC Control of pro apoptotic proteins under normal cell conditions of non apoptotic cells is incompletely understood Mitochondria are an important site for apoptosis Mitochondrial proteins known as SMACs second mitochondria derived activator of caspases are released into the cytosol following an increase in permeability SMAC binds to inhibitor of apoptosis prot
16. ure correct preparation Ensure sufficient incubation Time Incubate sample overnight Inadequate incubation times Low protein concentration Don t make too low dilution in sample Concentrate sample t kit Store kit at recommended mproper storage of ki temperatur High Excessive biotinylated Make sure correct Background antibodies amount of antibodies Excessive streptavidin Make sure correct amount ofstreptavidin Inadequate detection Check laser power Check PMT parameters Unclean environment such as dust Work in clean environment Increase wash time Use more wash buffer Insufficient wash Bubbles formed during Avoid bubble formation incubation during incubation Arrays are not completed Completely cover arrays Covered by reagent with solution RayBio Human Apoptosis Antibody Array G series 16 y pop VII Reference List 1 Zaparta JM Krajewska M Krajewski S Huang RP Takayama S Wang HG Adamson E and Reed JR 1998 Expression of multiple apoptosis regualtory genes in human breast cancer cell lines and primary tumors Breast Can Res amp Treat 47 129 140 2 Huang RP Huang R Fan Y and Lin Y 2001 A novel method for high throughput protein profiling from conditioned media and patient s sera Ana Biochem 294 1 55 62 3 Lue Jl and George DL 2007 Hepatic IGFBP1 is a prosurvival factor that binds to BAK protects the liver from apoptosis and antagonizes the proapoptotic actions of p53 at mitochon
17. ysates VI RayBio Analysis Tool RayBio Human Apoptosis Antibody Array G series 13 How to use RayBio Analysis Tool 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 eeee o 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 If you do not use our RayBio Analysis Tool you can locate the Apoptosis by referring to RayBio Human Apoptosis Antibody Array Normalization and comparison For biomarker discovery or for analysis of large number of arrays great attention must be paid to the normalization Our apoptosis antibody array design includes controls for normalization and comparison of arrays performing in different membranes and different experiments 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 normalizat
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