RayBiotech, Inc. RayBio® Human Apoptosis Antibody Array Kit
Contents
1. RayBio Human Apoptosis Antibody Array Kit User Manual Cat AAH APO 1 2 AAH APO 1 4 AAH APO 1 8 For Simultaneous Detection of the Relative Levels of 43 Apoptosis related Markers 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 RayBio Human Apoptosis Antibody Array Kit TABLE OF CONTENTS Ie WATOOUCTOM TEE 2 Il Principle of the 4 JE uz cm 6 a Material Provided 6 b Additional Materials Required 6 IV Reagent Preparation 7 V Overview and General Considerations 8 a Preparation of Samples 8 b Handling Array Membrane 8 8 WHE MEER 9 a Blocking and Incubation 9 b 10 VII Interpretation of Results 12 VIII Array 14 IX Troubleshooting Guide 15 X Reference List 16 RayBio Human Apoptosis Antibody Array Kit 1 I Introduction Apoptosis is the process of programmed cell death that involves a series of biochemical events leadin2. piece of plastic sheet Gently smooth out any air bubbles Avoid using pressure on the membrane 3 Detect signal directly from membrane using chemiluminescence imaging system or expose to x ray film we recommend to use Kodak X Omat AR film detect signal using film developer Expose the membranes for 40 Seconds 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 RayBio Human Apoptosis Antibody Array Kit 11 eg 2 20 min or overnight Or re incubate membranes overnight with 1X HRP conjugated Streptavidin and repeat detection on the second day 4 Save membranes at 20 C to 80 C for future reference VII Interpretation of Results The following figures show RayBio Human Apoptosis Antibody Array membranes probed with different cell lines The signals were detected by a chemiluminescene imaging device Membranes also can be exposed to Kodak X Omat film at room temperature A biotinylated protein provides positive signals which can be used to identify the orientation and to normalize the results from different wells being compared By comparing the signal intensities relative expression levels of target proteins can be made The intensities of signals can be quantified by densitometry Positive control can be used to normalize the results from different membr
3. 9 140 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 RayBiotech Inc the protein array pioneer company strives to research and develop new products to meet demands of the biomedical community RayBio patent pending technology allows detection of over 500 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 EIA kits Phosphotyrosine kits Recombinant Proteins Antibodies and custom services RayBio Human Apoptosis Antibody Array Kit 18 This product is for research use only 2009 RayBiotech Inc RayBio Human Apoptosis Antibody Array Kit 19
4. Array Kit 16 IX Troubleshooting Guide Weak signal or 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 HRP Conjugate streptavidin well before Streptavidin well before use since use precipitates may form during storage 4 Sample is too dilute 4 Increase sample concentration Reduce blocking concentration by diluting in 1X Wash Buffer II 5 Other 2 Slightly increase HRP concentrations 3 Slightly increase biotinylate antibody concentrations 4 Expose film for overnight to detect weak signal Bubbles formed Remove bubbles 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 Human Apoptosis Antibody Array Kit 17 X 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 regulatory genes in human breast cancer cell lines and primary tumors Breast Can Res amp Treat 47 12
5. MAC 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 caspases cysteine aspartic acid proteases which carry out the degradation of the cell Therefore the degradative activity of caspases seems 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 RayBio Human Apoptosis Antibody Array Kit 3 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 repair however it will induce apoptosis if damage is extensive and repair efforts fail Any disruption to th
6. anes After extensive washing the membranes are incubated with a cocktail of biotin conjugated anti apoptotic protein antibodies After incubation RayBio Human Apoptosis Antibody Array Kit 4 with HRP Streptavidin the signals visualized by chemiluminescence How It Works YYYYY Antibody array See Y chips Incubation of Sample with a vernigh Y Y Y Y d arrayed antibody chips Overnight p Cocktail of biotin conjugated anti gt apoptosis marker Incubation with cocktail of biotin conjugated anti apoptosis marker ry 7 hi Data analysis Overnight Labeled Streptavidin Incubation with labeled 2 hrs Streptavidin iit RayBio Human Apoptosis Antibody Array Kit 5 Illa Materials Provided Upon receipt the kit should be stored at 20 C After initial use 2X Cell Lysis Buffer Blocking Buffer 20X Wash Buffer 20X Wash Buffer l Cocktail of Biotin Conjugated antibody mix and HRP Conjugated Streptavidin should be stored at 4 C to avoid repeated freeze thaw cycles Array membrane Protease Inhibitor Cocktail should be kept at 20 C Please use the entire kit within 6 months from the date of shipment 1 2 RayBio Human Apoptosis Antibody Array 1 membranes 2 4 or 8 membranes 2X Cell Lysis Buffer 5 ml Protease Inhibitor Cocktail 1 tube for 2 4 membranes and 2 for 8 membranes 1X Blocking Buffer 25 ml for 2 4 membranes and 50 ml
7. anes being compared Antibody affinity to its target varies significantly between antibodies The intensity detected on the array with each antibody depends on this affinity therefore signal intensity comparison can be performed only within the same antibody antigen system and not between different antibodies One important parameter is the baseline signal response 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 RayBio Human Apoptosis Antibody Array Kit 12 1 Apoptotic protein profiling in selected cancer cell lines Bad bax bcl 2 Bcl w bid bim cas3 cas8 cIAP2 cytoC HTRA XI Fig 1 Apoptotic protein profiling in HeLa cell lines 400 ug of cell lysates from human MD231 BT549 and HeLa cell line were incubated overnight with RayBio Human Apoptosis Antibody Array membrane Control membrane was incubated with blocking buffer The antibody array membranes were then washed and cocktail of biotinylated antibody mix was used to detect apoptosis related proteins After incubation with HRP Conjugated Streptavidin the signals were visualized after 5 minute exposure by chemiluminescene RayBio Human Apoptosis Antibody Array Kit 13 2 Apoptotic protein profiling in induced Jurkat cell lines KT A Untreated Jurkat c
8. e 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 Il Principle of the Assay With RayBio Human Apoptosis Antibody Array kit researchers can now simultaneously detect the relative level of 43 apoptosis related proteins in cell lysates By monitoring the changes in protein levels in different experimental model systems researchers can study pathway activation without spending excess time and effort in performing immunoprecipitations and or Western Blotting With RayBio amp Human Apoptosis Antibody Array kit each of the capture antibodies is printed on the membranes and then treated or untreated cell lysate is added into antibody array membr
9. ells Bad bax bcl 2 Bcl w bid bim cas3 cas B Induced Jurkat cells cIAP2 cytoC HTRA In bold font Increase after induction In underline font decrease after induction In regular font no change after induction Livin XIAP Smac Survivin Fig 2 Apoptotic protein profiling in Induced and uninduced Jurkat cell lines Jurkat cells were treated with apoptosis inducer set for 7 hours 10mM Actinomycin 2mM Camptothecin 100mM Cycloheximide 10mM Dexamethasone and 100mM Etopiside 500 ug of cell lysate from both induced and uninduced Jurkat cells were incubated overnight with RayBio Human Apoptosis Antibody Array membrane Control membrane was incubated with blocking buffer The antibody array membranes were then washed and cocktail of biotinylated antibody mix was used to detect apoptosis related proteins After incubation with HRP Conjugated Streptavidin the signals were visualized after 5 minute exposure by chemiluminescene RayBio Human Apoptosis Antibody Array Kit 14 3 Apoptotic protein profiling in induced MCF 7 cell lines ue Untreated MCF 7 cells Bad bax bcl 2 Bcl w bid bim cas3 cas8 Induced MCF 7cells cIAP2 cytoC HTRA Livin XIAP In bold font Increase after induction In underline font decrease after induction Smac Survivin In regular font no change after induction Fig 3 Apoptotic protein profiling in Induced and uninduced MCF 7 cell lines MCF 7 cells were treated with apopto
10. extracts to microfuge tubes and centrifuge at 14 000 x g for 10 min It is recommended that sample protein concentrations be determined using a total protein assay such as Bradford Assay Before applying to the membranes all lysates should be diluted at least 5 fold with 1X Blocking Buffer For the RayBio Human Apoptosis Antibody Array Kit 200 ug ml to 600 ug ml of total protein from cell lysates after dilution with Blocking Buffer should be used for incubation Lysates should be used immediately or aliquotted and stored at 70 Thawed lysates should be kept on ice prior to use If you experience high background you may further dilute your samples B Handling Array Membranes RayBio Human Apoptosis Antibody Array Kit 8 e Always use forceps to handle membranes and grip the membranes by the edges only Flat tipped forceps are best for handling membranes e Never allow array membranes to dry during experiments e Avoid touching array membrane by hand tips or any sharp tools C Incubation e Completely cover membranes with sample or buffer during incubation and cover eight well tray with lid to avoid drying e Avoid foaming during incubation steps e Perform all incubation and wash steps under gentle shaking 1 2 cycles sec e All wash steps and several incubation steps such as step 3 sample incubation or step 7 biotin Ab incubation or step 10 HRP streptavidin incubation may be done overnight at 4 C VI P
11. for 8 membranes 20X Wash Buffer 10 ml for 2 4 membranes and 20ml for 8 membranes 20X Wash Buffer II 10 ml for 2 4 membranes and 20ml for 8 membranes Cocktail of Biotin Conjugated Antibody Mix 1 vial for 2 membranes 2 for 4 membranes and 4 for 8 membranes 1 000X HRP Conjugated Streptavidin I vial Detection Buffer C 1 5 ml for 2 4 membranes 2 5 ml for 8 membranes Detection Buffer D 1 5 ml for 2 4 membranes 2 5 ml for 8 membranes Eight Well Tray 1 each Plastic sheets Manual RayBio Human Apoptosis Antibody Array Kit 6 Additional Materials Required e Small plastic boxes or containers e Shaker e Plastic sheet protector or Saran Wrap e Kodak X Omat AR film REF 165 1454 and film processor or Chemiluminescence imaging system IV Reagent Preparation 1 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 20X Wash Buffer or Il the Wash Buffer Concentrate 20X contains visible crystals warm to room temperature and mix gently until dissolved D
12. g to characteristic cell morphology and death These events include blebbing changes to the cell membrane such as loss of membrane asymmetry and attachment cell shrinkage nuclear fragmentation chromatin condensation and chromosomal DNA fragmentation Studies on apoptosis 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 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 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 a
13. ilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1X Wash Buffer Cocktail of Antibody Mix Briefly spin the Biotin Conjugated Antibody tube before use Add 150 ul of Blocking Buffer to the tube Mix gently and transfer all mixture to a tube containing 1 80 ml of Blocking Buffer to prepare 1X Cocktail of Biotin Conjugated Antibody Mix 1000X HRP Conjugated Streptavidin briefly spin down the HRP Streptavidin Concentrate and pipette up and down to mix gently before use Prepare 1X HRP Conjugated Streptavidin Example RayBio Human Apoptosis Antibody Array Kit 7 add 5 ul of HRP Conjugated Streptavidin concentrate into a tube with 5 ml Blocking Buffer and mix gently Do not store the 1X Streptavidin for next day use Note mix tube containing 1 000X HRP Conjugated Streptavidin well before use since precipitation may form during storage V Overview and General Considerations A Preparation of Samples Remove supernatant from cell culture for attached cells wash cells twice with cold 1X PBS for suspension cells pellet the cells by spinning down the cells at 1500 rpm for 10 min making sure to remove any remaining PBS before adding Lysis Buffer Solubilize the cells at 2x10 cells ml in 1X Lysis Buffer containing Protease Inhibitor Cocktail prepared in step 2 of Reagent Preparation Pipette up and down to resuspend cells and rock the lysates gently at 2 8 C for 30 minutes Transfer
14. nd 2 The binding of TNF to 1 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 RayBio Human Apoptosis Antibody Array Kit 2 cell survival and inflammatory responses The Fas receptor also known as 1 or CD95 binds the Fas ligand The interaction 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 2 members of the 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 S
15. on may be done at room temperature for 2 hours 8 Wash as directed in steps 4 5 and 6 9 Add 1 5 ml of 1X HRP conjugated Streptavidin to each membrane Note Mix tube containing 1X HRP Conjugated Streptavidin well before use since precipitation may form during storage 10 Incubate at room temperature for 1 5 hours RayBio Human Apoptosis Antibody Array Kit 10 Note incubation may be done overnight at 4 C 12 Wash as directed in steps 5 and 6 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 detection reaction Add 250 ul of Detection Buffer C and 250 ul of 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 on to the membrane and incubate at room temperature with gentle shaking for 2 minutes Ensure that the detection mixture is completely and evenly covering the membrane without any air bubbles 2 Drain off 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 the array with another
16. rotocol A Blocking and Incubation 1 Place each membrane into the provided 8 well tray top left corner marked with Note The printed side should be facing upward 2 Add 1 ml Blocking Buffer and incubate at room temperature with gentle shaking for 30 minutes to block membranes 3 Decant Blocking Buffer from each well Add 1 ml diluted sample onto each array membrane and cover with the lid Incubate at 2 8 C overnight on a rocker or shaker low speed 1 2 cycles sec Dilute sample using Blocking Buffer RayBio Human Apoptosis Antibody Array Kit 9 Note 1 Dilute cell lysates at least 5 folds with Blocking Buffer to avoid high background Note 2 Optimal sample dilution factors should be determined empirically More of the sample can be used if signals are too weak If signals are too strong the sample can be diluted further Note 3 Incubation may be done at room temperature for 4 hours but at the cost of impaired sensitivity of the signals 4 Decant the samples from each well and wash 3 times with 1 5 ml of 1X Wash Buffer at room temperature with shaking 5 min per wash 5 Wash 2 times with 1 5 ml of 1X Wash Buffer at room temperature with shaking 5 min per wash 6 Carefully remove wash buffer from each well containing array membranes 7 Add 1 ml of diluted Cocktail of Biotin Conjugated Antibody Mix to each membrane Incubate at 2 8 with gentle shaking overnight Note Incubati
17. sis inducer set for 7 hours 10mM Actinomycin D 2mM Camptothecin 100mM Cycloheximide 10mM Dexamethasone and 100mM Etopiside 500 ug of cell lysate from both induced and uninduced MCF 7 cells were incubated overnight with RayBio Human Apoptosis Antibody Array membrane Control membrane was incubated with blocking buffer The antibody array membranes were then washed and cocktail of biotinylated antibody mix was used to detect apoptosis related proteins After incubation with HRP Conjugated Streptavidin the signals were visualized after 5 minute exposure by chemiluminescence RayBio Human Apoptosis Antibody Array Kit 15 Vill Human Apoptosis Antibody Array Map L N bck2 bew Caspase3 caspases bad bax 52 bow Bim Caspase3 caspases Fas Fast blank 5 27 HsPeo Hsp7o Gri s coo cape Fas FasL blank HSP27 nseeo WSP7O HTRA ieri icrBP 2 icrBP 3 4 5 IGFBP 6 iGF isR livin p27 SMAC Survivin sTNF R1 5 Ls eren erm ima pe oso T sment 7 TNF alpha TNF beta TRAILR 1 manna TRALR S maea Xa Blank Blank Neg Neg e sre TNFalpha TNF beta TRALR2 ratas TRALR Blank Blank Neg neg RayBio Human Apoptosis Antibody
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