CASE Kit User Manual
Contents
1. Cell Staining Solution Developing Solution Stop Solution Washing Buffer Reservoir 4 Plate Sealing Film 4 Anti Phospho Protein Specific Antibody Anti Pan Protein Specific Antibody Secondary Antibody 10X Washing Buffer must be diluted to 1X with distilled H2O before use Each antibody stock must be freshly diluted into Antibody Dilution Buffer on the same day of the experiment Use Dilution Factor specified for each antibody on its Product Information sheet See page 9 for more details Just before use dissolve powder in 30 ml 1X Washing Buffer by mixing well Once dissolved Blocking Buffer may be stored at 4 C for up to two 2 weeks Shipping and Storage Conditions Box 1 is shipped at ambient temperature and should be stored at 4 C Box 2 is shipped on dry ice and should be stored at 20 C Shelf Life All reagents are stable as is if it is not used for 6 months after receipt of the kit and stored at the recommended temperature SuperArray CASE Kit User Manual Version 2 0 5 25 2005 8 Ill Additional Materials Required The following solutions are required for performing the CASE assay but are not included with the kit These solutions must be prepared from materials obtained from other manufacturers before starting the CASE procedure 1X Phosphate Buffered Saline PBS Dissolve 8 0 g of NaCl 0 2 g of KCI 1 44 g of NagHPQ and 0 24 g of KH2PO in 800 ml of distilled HzO Adjust the2. Profiling Pathway Activation via Cell Based Detection of Protein Phosphorylation BIOMOLGmbH Waidmannsr 35 A 22769 Ha mburg O biomol info biomol de www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D Technical Support Phone 49 40 853260 23 27 37 ts biomol de
3. Secondary antibody concentration is too high Decrease the secondary antibody concentration to half or reduce the secondary antibody incubation time from 1 h to 30 min F High absorbance in blank wells Stop Solution was contaminated with Developing Solution Be sure to use a new and clean Buffer Reservoir for Stop Solution Do not use a Buffer Reservoir that has been previously used to contain Developing Solution to hold Stop Solution G Significant signal variation among replicate wells 1 Cell loss during incubation and washing Be sure to normalize antibody signals with the cell staining reading Asgs A well with an As 5 reading close to the reading of a blank well lacking cells indicates a complete loss of cells from that well Omit this well from your data set Improper antibody incubation and washing Be sure to fill all wells with the appropriate antibody and follow the recommendations for washing Use a rocker in every wash step H No antibody solution in wells after overnight incubation Be sure to seal the open plate with sealing tape then with a piece of parafilm and place the lid onto the plate The plate may also be stored in a zip lock or heat sealed bag Be sure to store the plate on a level stable surface SuperArray CASE Kit User Manual Version 2 0 5 25 2005 This page is intentionally left blank SuperArray CASE Kit User Manual Version 2 0 5 25 2005 20 Cellular Activation of Signaling ELISA
4. Reader If the signals are greater than the linear range of the instrument remove 50 ul of the 1 SDS solution from every well and add 50 ul of dH20 to each well Repeat the reading of the absorbance at 595 nm on the ELISA Plate Reader Normalize the antibody reading to the relative cell number Divide the OD so readings for each well by its ODsg5 reading To determine the relative extent of target protein phosphorylation normalize the phospho protein specific antibody ODy459 0Ds95 ratio to the pan protein specific antibody OD 4s50 ODs95 ratio for the same experimental condition SuperArray CASE Kit User Manual Version 2 0 5 25 2005 17 V Troubleshooting Guide A No or weak signal in wells incubated with either the phospho protein specific or the pan protein specific primary antibody 1 Improper Plate Reader settings Verify the wavelength and filter settings of the ELISA Plate Reader Cold Developing Solution Warm the Developing Solution to room temperature before use Insufficient numbers of cells remain on the plate Check cells on the plate using microscope and perform steps to Determine Relative Cell Number to measure how many cells remain on the plate Cells do not contain detectable levels of the target protein Verify expression of the protein in your cell line by Western analysis using a pan protein specific antibody B No response observed to experimental treatments in wells incubated with phospho protein
5. pH to 7 4 Bring the final volume to 1 liter with distilled water Dispense in convenient volumes and sterilize by autoclaving Store at room temperature 37 Formaldehyde Molecular Biology Grade from any chemical supplier For example Sigma Cat No F8775 30 H202 concentrated hydrogen peroxide Molecular Biology Grade from any chemical supplier For example Sigma Cat No H1009 10 NaN sodium azide Dissolve 10 g of sodium azide into 100 ml of ddH20 Use appropriate caution sodium azide is a hazardous substance 1 SDS sodium dodecyl sulfate Dissolve 1 g of sodium dodecyl sulfate into 100 ml of ddH20 Store at room temperature 0 01 poly L Lysine for suspension cells onl Molecular Biology Grade from any chemical supplier For example Sigma Cat No P4832 The following supplies are also required but are not included in the kit and must be purchased separately 96 well Tissue Culture Plates for example Corning Costar 3595 Multi channel Pipettor Parafilm Rocking platform ELISA Plate Reader capable of reading at 450 and 595 nm SuperArray CASE Kit User Manual Version 2 0 5 25 2005 9 IV Protocol PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING EXPERIMENT A Working Solution Preparation The following working solutions should be freshly prepared from the stock solutions each time the experiment is performed using the following recipes 1 Cell Fixing Buffer Prepare either 4 or 8 Cell
6. specific antibody to each appropriate well See page 9 for antibody dilution instructions To the negative control wells add 50 ul of Antibody Dilution Buffer instead To prevent evaporation use a piece of the Plate Sealing Film provided in kit followed by a piece of parafilm to seal the plate Then cover the plate with its lid Incubate overnight at 4 C Remove the primary antibody and wash cells twice with 200 ul of 1X Washing Buffer for 5 minutes each with rocking Remove the Washing Buffer and add 100 ul of dilute secondary antibody to each well Incubate for 1 hour at room temperature Meanwhile transfer only the needed amount of Developing Solution 100 ul per well into a clean Washing Buffer Reservoir and allow it to warm to room temperature while protected from light SuperArray CASE Kit User Manual Version 2 0 5 25 2005 15 D Colorimetric Detection 1 Remove the secondary antibody and wash cells twice with 200 ul of 1X Washing Buffer for 5 minutes each with rocking Then wash the cells once with 200 ul of PBS for 5 minutes with rocking Remove the PBS and add 100 ul of Developing Solution pre warmed to room temperature into each well Protect plate from direct light Incubate for 2 to 20 minutes at room temperature Monitor the blue color development until the darkest staining well turns a medium to dark blue DO NOT OVERDEVELOP ANY WELLS Add 100 ul Stop Solution into each well
7. 4 C to 2 h at 37 C C Poor sensitivity low absorbance readings for antibodies Primary antibody concentration is too low For low protein expression levels as determined by Western analysis double the concentration of the primary antibodies and decrease the incubation volume by half Take extra care in dispensing the smaller volumes and in sealing the plate to avoid evaporation SuperArray CASE Kit User Manual Version 2 0 5 25 2005 18 D The Aso readings for the wells incubated with the phospho protein specific antibody are higher than the A459 readings from wells incubated with the pan protein specific antibody The phospho protein specific antibody is simply more sensitive than that the pan protein specific antibody This phenomenon occurs in several of our CASE Kits It does not affect your final assay results Remember that the CASE Kits do not involve a calibration curve and therefore can only be used for relative measurement E High background in all wells 1 Developing time too long Be sure to stop the colorimetric development as soon as the darkest positive wells turn a medium to dark blue Improper blocking and washing Be sure that washing quenching and blocking steps were all performed according to the recommendations in the protocol Remember to add the solutions carefully down the sides of the wells and to remove liquids by inverting plates over a sink and then tapping the plate gently on a paper towel
8. A SuperArray Waidmannstr 35 A Bioscience Corporation 22769 Ha mburg C biomol info biomol de www biomol de U ser M anu al Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D Part 1013A Cellular Activation of Signaling ELISA Profiling Pathway Activation via Cell Based Detection of Protein Phosphorylation See Purchaser Notification for limited use license and warranty information page 3 SuperArray CASE Kit User Manual Version 2 0 5 25 2005 This page is intentionally left blank SuperArray CASE Kit User Manual Version 2 0 5 25 2005 3 Cellular Activation of Signaling ELISA CASE Kit Profiling Pathway Activation Via Cell Based Detection of Protein Phosphorylation USER MANUAL ORDERING INFORMATION AND TECHNICAL SERVICE e TEL 0800 2466651 D 49 40 8532600 e FAX 0800 2466652 D 49 40 85326022 e ON LINE ORDER www biomol de e E MAIL info biomol de to place an order ts biomol de for technical support You may place orders by phone fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information MasterCard Shipping address Billing address For more information visit us at http www biomol de SuperArray Bioscience 7320 Executive Way Suite 101 Frederick MD 21704 USA SuperArra
9. F 1 0 1 10 100 10 ng ml IGF 1 VY YYVYYY NI OOOO 5 _ gt OK PK eR RN ON 5 Anti Phospho Protein ihe a Specific Antibody g E E 5 i Anti Pan Protein E 3 Specific Antibody g F EA Figure 2 Example of how to plate cells for AKT CASE Kit Seed cells and plan treatment in two identical sets of cell culture wells In the example here a 96 well plate is divided into two sets and treatments are identical in both sets in a symmetrical fashion Duplicate wells are used here for each dose and time point Cells were treated with various concentrations 0 1 10 or 100 ng ml of IGF 1 with or without the kinase inhibitor LY294002 for 0 10 30 or 60 minutes The 96 well plate was used in a CASE Kit Assay half of the plate was treated with the anti phospho protein specific primary antibody and half with the anti pan protein specific primary antibody Eight wells of cells were not treated with primary antibody but only with secondary antibody 2 Antibody ONLY and eight wells were not plated with cells but were treated with both primary and second antibodies Blank The different shades in 96 well plate represent theoretical signal intensities from the experiment a Each treatment point or assay determination requires two sets of cells treated in an identical fashion One set of cells will be incubated with the phospho protein specific antibody to measure phosphorylated protein the other set of cells will b
10. Fixing buffer depending on the type of your cell culture Volume for 96 Cell Fixing Buffer Reagent wells F 1X PBS 10 7 ml 0 ea 37 Formaldehyde 1 3 ml Total Volume 12 ml OR 1X PBS 9 4 ml o eal Rel en 37 Formaldehyde 2 6 ml p Total Volume 12 ml 2 1X Washing Buffer 10X Washing Buffer must be diluted to 1X with distilled H20 before use 3 Quenching Buffer To 11 48 ml of 1X Washing Buffer add 400 ul of 30 H202 and 120 ul of 10 NaNs for a final volume of 12 ml enough for 96 wells 4 Blocking Buffer Just before use dissolve blocking buffer powder provided in kit box 1 in 30 ml 1X Washing Buffer by mixing well Once dissolved Blocking Buffer may be stored at 4 C for up to two 2 weeks SuperArray CASE Kit User Manual Version 2 0 5 25 2005 10 5 Antibody Dilution Each antibody provided in the kit must be freshly diluted using Antibody Dilution Buffer each time the experiment is performed The dilution factor for each antibody is specified on each kit s product information sheet Volume of Diluted Antibody Prepared in Antibody Dilution Buffer Diluted Antibody 8 assays 12 assays 96 assays SOE D AE A 500p 750 ul 6000 pl a Soom so evou Secondary Antibody 2000 ul 3000 ul 24000 ul Use the appropriate dilution factor for each antibody listed on the individual kit product information sheet For example If the required dilution factor for an anti phospho protein specific antibody
11. action electrophoresis or Western blot Detect relative amount of total and phosphorylated target protein as well as cell number all at the same time Method Reference Versteeg HH Nijhuis E van den Brink GR Evertzen M Pynaert GN van Deventer SJ Coffer PJ Peppelenbosch MP A new phosphospecific cell based ELISA for p42 p44 mitogen activated protein kinase MAPK p38 MAPK protein kinase B and cAMP response element binding protein Biochem J 2000 Sep 15 350 Pt 3 717 22 SuperArray CASE Kit User Manual Version 2 0 5 25 2005 6 CASE Kit Quick Reference Procedure Assay time lt 3 hours of hands on time Plate and Treat Cells Fix Cells Wash Quench Wash 1 5 hours Block Wash Incubate with Primary Antibodies Phospho Protein Specific Pan Protein Specific Wash Incubate with HRP Conjugated Seconday Antibody Wash Overnight 4 2C One hour room temperature Develop Colorimetry Read OD lt 9 Wash Stain Cells Wash Read ODz9s 10 20 minutes Figure 1 Overview of Cellular Activation of Signaling ELISA CASE Kit Procedure SuperArray CASE Kit User Manual ll Materials Provided Version 2 0 5 25 2005 7 This kit includes enough of the following reagents for 96 assays Box 1 Assay and Detection Reagents Store at 4 C COMPONENTS Box 2 Antibodies Store at 20 C COMPONENTS 10X Washing Buffer Antibody Dilution Buffer Blocking Buffer dry powder
12. cell culture wells without sacrificing specificity reproducibility or sensitivity With its 96 well plate format the CASE assay requires very small amounts of cells and treatment reagents and the assay procedure is compatible with high throughput studies These features make the CASE kits especially suitable for screening compounds that activate or inhibit important signaling pathways In the CASE assay cells are seeded into 96 well plates After your experimental treatment the cells are fixed to preserve any activation specific protein modification such as phosphorylation Two primary antibodies are included in the kit One antibody recognizes only the activated phosphorylated form of the specific target protein while another recognizes the specific target protein regardless of its activation state Following incubation with primary and secondary antibodies the amount of bound antibody in each well is determined using a developing solution and an ELISA Plate Reader The absorbance readings are then normalized to relative cell number as determined by a cell staining solution The amount of phosphorylated protein once normalized to the amount of total protein is then directly related to the extent of downstream pathway activation Features and Advantages of the CASE Kits Easy quantitative and non radioactive protocol with lt 3 hours hands on time No loss of activation state during procedure Cell based assay No cell extr
13. ding to your institution s hazardous waste disposal protocol You may use the fixed cells right away or the fixed cells are also stable for several weeks in Cell Fixing Buffer Seal with parafilm or in a zip lock bag and refrigerate at 4 C In this way you can accumulate many plates in the refrigerator and perform the CASE assays later when desired NOTE To minimize cell loss during all wash steps in the following protocol never dispense liquid directly onto the cell surface Instead gently dispense liquid down the wall of cell culture wells To remove wash solutions flip the plate over a sink and then tap the plate gently onto a paper towel to remove any remaining liquid 4 Remove the Cell Fixing Buffer and wash cells twice with 200 ul of 1X Washing Buffer for 5 minutes each with rocking 5 Remove Washing Buffer add 100 ul of Quenching Buffer and incubate for 20 minutes at room temperature 6 Remove Quenching Buffer and wash cells once with 200 ul of 1X Washing Buffer for 5 minutes with rocking 7 Remove Washing Buffer add 100 ul of Blocking Buffer and incubate 1 hour at room temperature SuperArray CASE Kit User Manual Version 2 0 5 25 2005 14 C Incubation with Primary and Secondary Antibodies 1 Remove Blocking Buffer and wash cells once with 200 ul of 1X Washing Buffer for 5 minutes with rocking Remove Washing Buffer and add 50 ul of diluted primary antibody either the phospho protein or the pan protein
14. e incubated with the pan protein specific antibody to measure total protein Each experimental condition e g time point concentration point etc should be performed in duplicate or triplicate to control for systematic variation SuperArray CASE Kit User Manual Version 2 0 5 25 2005 12 b You should also include wells for control purposes such as i Blank wells not seeded with any cells ii Detection control wells seeded with cells only incubated with secondary but not primary antibody iii Experimental control wells seeded with cells but not experimentally treated NOTE If using non adherent that is suspension cells pre coat tissue culture wells by filling them with 10 ug ml poly L Lysine incubating at 37 C for 30 minutes and washing twice for 5 minutes with 1X PBS c Seed cells into a 96 well plate the day before your treatment with the idea that the cell culture density should be at roughly 50 80 percent confluence on the day of your treatment and the CASE experiment For example In the experiment outlined in Figure 2 cells are seeded at 2 0 x 10 cells per well on day 0 and allowed to sit and grow overnight On day 1 the cells are starved in serum free medium overnight On day 2 the relevant wells of cells are pre treated with the inhibitor for one hour followed by the IGF1 treatment as described in the figure legend Then the CASE assay protocol is performed as described below For the majority of cell line
15. is 1 150 and 12 assays are to be performed calculate the amount of stock antibody to dilute 750 ul from table above 150 dilution factor 5 ul Thus 5 ul of antibody should be diluted into 750 ul of Antibody Dilution Buffer Perform this calculation separately for both primary antibodies and the secondary antibody using their dilution factors and the total volumes in the table above Dilute just enough of the antibodies as needed for the planned experiment If you use the antibodies wisely enough material is provided in the kit to process a total of 96 assays or in other words 96 wells with each primary antibody and a total of 2 x 96 192 wells with the secondary antibody SuperArray CASE Kit User Manual Version 2 0 5 25 2005 11 B Cell Culture and Cell Preparation NOTE A fresh and healthily growing cell culture is crucial for a successful CASE experiment Only use cells growing in log phase to set up a CASE experiment If the cell culture is already too old that is if the cells are over confluent if the culture medium turns yellow in color or if adherent cells have started floating to a significant extent split the cells once into a fresh tissue culture flask or dish and fresh medium Allow the cells to grow into log phase once more before seeding into a 96 well plate to set up the CASE experiment 1 Plan your experiment and your cell culture carefully For example see Figure 2 LY294002 5 LY294002 ng ml IG
16. s with a doubling time of 18 24 h seeding an initial density of roughly 1 5 x 104 cells per well will yield the appropriate cell density by the time of the CASE assay If you have not established a growth curve for your cell line in 96 well tissue culture plates perform a pilot cell seeding experiment before seeding cells for the CASE experiment Cell densities that are too high or too low at the time of the CASE assay give poor results Re seed cells if the cell growth or confluence conditions are not appropriate NOTE It is not necessary to plate cells in all 96 wells Only add cells to the wells that you plan to use and prepare only as much antibody dilution as you need SuperArray CASE Kit User Manual Version 2 0 5 25 2005 13 2 Allow cells to grow overnight Then perform treatments as defined by your experiment on the next day 3 Fix Cells a For adherent cells remove the culture medium and add 100 ul of 4 Cell Fixing Buffer to each well b For suspension cells directly add 100 ul of 8 Cell Fixing Buffer to the 100 ul of culture medium in each well To minimize formaldehyde evaporation seal the open plate with a piece of parafilm and place the lid onto the plate Incubate for 20 minutes at room temperature WARNING Formaldehyde and its vapors are highly toxic Always prepare and use formaldehyde solutions under a chemical fume hood and wear protective gloves and eyewear and a lab coat Dispose formaldehyde waste accor
17. specific antibody 1 Cells culture conditions are not appropriate When cells are not healthy i e the cell culture is not fresh or the cell density is too low or over confluent cells may not respond to your treatment properly Seed cells the day before your treatment so that the cell culture density is at roughly 50 80 percent confluence before your treatment If you have not established a growth curve for your cell line in 96 well tissue culture plates perform a pilot cell seeding experiment before seeding cells for the CASE experiment For the majority of cell lines with a doubling time of 18 24 h seeding an initial density of roughly 1 5 X104 cells per well will yield the appropriate cell density by the time of the CASE assay Treatment does not activate target protein By Western analysis using a phospho protein specific antibody verify that your treatment conditions are actually activating the target protein Use a well established treatment known to induce the specific protein phosphorylation event as your positive control Insufficient antibody sensitivity If the absorbance readings in the wells incubated with phospho protein specific antibody are significantly above background but do not change in response to your treatment then the ability to detect smaller changes can be improved by decreasing the concentration of anti phospho protein specific antibody to half or reducing the antibody incubation time from overnight at
18. ssed implied or by estoppel is granted 11 14 15 16 17 SuperArray CASE Kit User Manual Version 2 0 5 25 2005 5 I BACKGROUND AND INTRODUCTION As an intracellular signal transduction pathway involving a protein kinase cascade is activated by an extracellular stimulus the extent of phosphorylation of an upstream regulatory protein increases Monitoring the phosphorylation status of this protein helps verify whether the stimulus activates the pathway and helps determine the effect of various treatments inhibitors or activators on that activation Two traditional methods are widely used to measure the phosphorylation status of a protein Western blot analysis with phospho specific antibody or P incorporation followed by gel electrophoresis Both methods are tedious require a large amount of cells and treatment reagents and are not suitable for high throughput studies The Cellular Activation of Signaling ELISA CASE Kits employ a cell based ELISA that directly measures protein phosphorylation on 96 well cultured cells The CASE Kits include a complete antibody based detection system for colorimetric quantification of the relative amount of phosphorylated protein and total target protein This simple and efficient cell based protein phosphorylation assay eliminates the need to prepare cell lysates and to perform a Western blot or the need to use r 2P ATP for in vitro kinase assays The entire assay occurs directly in
19. to stop reaction The Stop Solution turns the blue color to yellow Note Do not add Stop Solution to the Washing Buffer Reservoir that contained the Developing Solution Use a clean reservoir for Stop Solution Stagger the addition of the Developing and Stop Solutions in a manner that allows each well to develop for the same length of time WARNING The Stop Solution is corrosive Always wear protective gloves and eyewear and a lab coat when handling this solution 5 Within 5 minutes read absorbance at 450 nm on an ELISA Plate Reader Blank the instrument according to the manufacturer s instructions SuperArray CASE Kit User Manual Version 2 0 5 25 2005 16 E Determine Relative Cell Number 1 2 Remove the assay solution as described above Wash the wells once with 200 ul of 1X Washing Buffer for 5 minutes with rocking Wash the wells once with 200 ul of dH20 for 5 minutes with rocking Be sure to remove any excess liquid by tapping plate gently on paper towel Air dry at room temperature for 5 minutes Add 100 ul of Cell Staining Buffer to each well and incubate for 30 minutes at room temperature WARNING The Cell Staining Buffer is an intense stain Avoid contact with skin and clothing 3 4 Wash the wells twice with 200 ul of dH20 for 5 minutes each with rocking Add 100 ul of 1 SDS to each well and incubate on a rocker for 1 hour at room temperature Read absorbance at 595 nm on an ELISA Plate
20. y CASE Kit User Manual Version 2 0 5 25 2005 TABLE OF CONTENTS l Background and Introduction ll Materials Provided Ill Additional Materials Required IV Protocol A Working Solution Preparation B Cell Culture and Cell Preparation C Incubation with Primary and Secondary Antibodies D Colorimetric Detection E Determine Relative Cell Number V Troubleshooting Guide LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SuperArray Bioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SuperArray Bioscience Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER The purchase of a CASE Kit includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any kit component to modify kit components for resale or to use a CASE Kit to manufacture commercial products without written approval of SuperArray Bioscience Corporation No other license expre
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