User Manual - RayBiotech, Inc.
Contents
1. Note Incubation may be done for 2 hours at room temperature 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 gt 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 II with H2O Completely remove wash buffer II in each wash step RayBio G Series Human Apoptosis Antibody Array 10 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 gt Note the diluted biotin conjugated antibodies can be stored at 4 C for 2 3 days 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 overnight at 4 C 9 Wash as directed in steps 5 and 6 10 Add 70 ul of 1 500 fold diluted HiLyte Plus conjugated streptavidin after brief spinning add 1 5 ml of Blocking Buffer to HiLyte Plus 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 hours2. V Array Map RayBio Human Apoptosis Antibody Array G series A B B D E F G H l J K L M o S Pos Pos Neg Neg ba bew B 7 ror Pos Pos neo Neg ba bolw BD CD40 CD40L clAP 2 cytoC DR6 Fas FasL neg HSP27 HSP60 HSP70 HTRA IGF I CD40 CD40L clAP 2 cytoC DR6 Fas FasL neg HSP27 HSP60 HSP70 HTRA IGF I os eem icrep icrep2 icrers icrer 4 icree s icrep iGrisk ivn p21 pz ps smc scr icrep icrep2 icrep s icrer 4 icree s icrep iGr isk vin pet pz ps smac 7 Survivin sTNF R1 sTNF R2 TNF alpha TNF beta TRAILR 1 TRAILR 2 TRAILR 3 TRAILR 4 XIAP Neg Neg Neg 8 Survivin sTNF R1 sTNF R2 TNF alpha TNF beta TRAILR 1 TRAILR 2 TRAILR 3 TRAILR 4 XIAP Neg Neg Neg 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 G Series Human Apoptosis Antibody Array 15 VI Troubleshooting guide Problem Cause Recommendation Weak signal Inadequate detection Check laser power Check PMT parameters Inadequate reagent volume Check pipettor Improper dilution Ensure correct preparation vida of Ensure sufficient incubation Inadequate incubation times Time Incubate sample overnight Low protein concentration Don t make too low dilution in sample Concentrate sample i f kit Store kit at recommended mproper storage of
3. a lll Overview and General Considerations A Preparation of Samples 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 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 5 10 fold with 1X Blocking Buffer for array assay 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 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 We recommend a working concentration of 500 600 ug ml of total protein for cell lysates and tissue lysates Since we RayBio G Series Human Apoptosis Antibody Array 7 recommend at least 5 10 fold dilution of lysate samples the original lysate concentration should be at least 2 5 mg ml The kit can also be used for serum and plasma samples as well as conditioned medium If you experience high ba
4. conjugated protein produces positive signals which can be used to identify the orientation and to compare the relative expression levels among the different wells RayBio G Series Human Apoptosis Antibody Array 12 Uninduced Jurkat Cell Induced Jurkat Cell Bax 2 Bcl 2 Bim Caspase 3 Cyto C OHR 7 HR ee Bcl 2 ae HTRA 2 Survivin XIAP Smac CAEN AreN 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 lysates RayBio G Series Human Apoptosis Antibody Array 13 VI RayBio Analysis Tool How to use RayBio Analysis Tool The signal intensities obtain
5. ki mnerat re Excessive biotinylated Make sure correct High 2 i antibodies amount of antibodies Background Excessive streptavidin Make sure correct amount of streptavidin Inadequate detection Check laser power Check PMT parameters Unclean environment such as dust Work in clean environment Insufficient wash Increase wash time Use more wash buffer Bubbles formed during Avoid bubble formation incubation during incubation Arrays are not completed Completely cover arrays Covered by reagent with solution RayBio G Series Human Apoptosis Antibody Array 16 Vil 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 mitochondria Genes amp Dev 21 3095 3109 4 Thompson CB 1995 Apoptosis in the pathogenesis and treatment of disease Science 267 5203 1456 62 5 Brune B 2003 Nitric oxide NO apoptosis or turning it ON Cell Death Differ 10 8 864 9 6 F
6. RayBio G Series Human Apoptosis Antibody Array User Manual Revised September 29 2015 Human Apoptosis Array G1 Cat AAH APO G1 4 Cat AAH APO G1 8 Please read manual carefully before starting experiment RayBiotech Inc We Provide You with Excellent Protein Array Systems and Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 1 888 547 0580 Website www raybiotech com Email info raybiotech com RayBiotech Inc Human Apoptosis Array G1 Protocol TABLE OF CONTENTS l MOGUC HO aneia stein terror cements FOW TOW OPK S ursina REEERE Il Materials Provided cccccccceeeeeeeeeeeeeees Additional Materials Required 55 Ill Overview and General Considerations A Preparation of Samples cceceeeees B Handling Glass Chips ceceese eee eees C Incubation s22c2 cen cnsotasendatnsaperawhore banennseceees No PROC CON tenes cn teaseretenciace tatneteweaekavasaeaetes A Blocking and Incubation 00ceeee eee B Delect pcssteescecosaesanreestnacoeqcoteeaesaceace V Interpretation of Results cccce cence ees VI RayBio Analysis TOol 0ccecceee eee eeaeeaes VIL Aray Map orcas anna A Eai VIII Troubleshooting Guide ccceeeeee eee eees IX Reference List ccccccce cece cease ease eeeees RayBio is the trademark of RayBiotech Inc RayBio G Series Human Apoptosis A
7. ckground 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 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 HiLyte Plus streptavidin incubation may be done at 4 C for overnight Please make sure to cover the incubation chamber tightly to prevent evaporation RayBio G Series Human Apoptosis Antibody Array 8 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 incubation frame as shown below Note if you slide has been pre assembled you can go to step 3 directly Instructions for incubation chamber asse
8. e 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 HiLyte Plus is the trademark of AnaSpec Inc GenePix is a registered trademark of Molecular Devices Inc RayBio G Series Human Apoptosis Antibody Array 18 This product is for research use only 2015 RayBiotech Inc RayBio G Series Human Apoptosis Antibody Array 19
9. econd mitochondria derived activator of caspases are released into the cytosol following an increase in permeability SMAC binds to inhibitor of apoptosis proteins 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 G Series Human Apoptosis Antibody Array 3 repair h
10. ed 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 io 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 normalization Negative control Negative control is BSA Normally it should only give a background reading RayBio G Series Human Apoptosis Antibody Array 14
11. es Human Apoptosis Antibody Array 5 ll Materials Provided Upon receipt all components of the RayBio Human Apoptosis Antibody Array kit should be stored at 20 C At 20 C the kit will retain full activity for up to 6 months Once thawed the glass chips HiLyte Plus streptavidin Proteinase Inhibitor and 1X Blocking Buffer should be kept at 20 C and all other components 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 or 8 subarray wells in one slide 2 Biotin Conjugated Anti Apoptosis Antibody cocktail mix 1 vial for 4 wells of each slide 2 vials for 8 wells of each slide 1 500X HiLyte Plus 555 Conjugated Streptavidin 1 vial 1X Blocking Buffer 10 ml 20X Wash Buffer 30ml 20X Wash Buffer II 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 2 ONO eee Additional Materials Required e Orbital shaker e Laser scanner for fluorescence detection e Aluminum foil e Distilled water e Plastic box RayBio G Series Human Apoptosis Antibody Array 6 Layout of RayBio Human Apoptosis Antibody Array Blank gt mee E E E OOOOOOO NOG OOS E L Barcod Barcod ALAA
12. esik 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 G Series Human Apoptosis Antibody Array 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 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 ar
13. gt 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 RayBio G Series Human Apoptosis Antibody Array 11 y P Repeat Wash Buffer 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 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
14. mbly G Series and Q series arrays Incubation chamber gt gt Gasket normally attached to chamber Protective Cover can ee be discarded CEEE Snap on sides ee 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 RayBio G Series Human Apoptosis Antibody Array 9 3 Add 100 ul 1X Blocking Buffer into each well and incubate at room temperature for 30 min to block slides 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 70 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 gt Note Dilute the lysate at least 10 fold with 1 X blocking buffer to make a total volume of 50 to 100 ul Make sure there are no bubbles in the wells gt 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 gt
15. nd mediated model TNF is the major extrinsic mediator of apoptosis Most cells in the human body have two receptors for TNF TNF R1 and TNF R2 The binding of TNF to TNF R7 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 7 or CD95 binds the Fas ligand The interaction RayBio G Series Human Apoptosis Antibody Array 2 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 R1 and Fas activation in mammalian cells a balance between pro apoptotic BAX BID BAK or BAD and anti apoptotic Bcl XI 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 s
16. ntibody Array 1 y P I Introduction Apoptosis is the process of programmed cell death that involves a series of biochemical events leading to a characteristic 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 liga
17. owever 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 impaired 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 G Series Human Apoptosis Antibody Array 4 Here s how it works Array a aX support Athn 6 Samples lt Incubation of Sample v YY With arrayed antibody Overnight Supports Cocktail of Biotin Ab KKAK t KAK Incubation with YYY Biotinylated Ab Labeled a da d da streptavidin OE Incubation with h fly S 2 hrs Labeled streptavidin ee gt Detection of signals Data analysis and graph RayBio G Seri
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