96-Well Ras Activation ELISA Kit (Chemiluminescent)
Contents
1. Product Manual 96 Well Ras Activation ELISA Kit Chemiluminescent Catalog Number STA 441 96 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC Creating Solutions for Life Science Research Introduction Small GTP binding proteins or GTPases are a family of proteins that serve as molecular regulators in signaling transduction pathways Ras a 21 kDa protein regulates a variety of biological response pathways that include cell growth cell transformation and tumor invasion Like other small GTPases Ras regulates molecular events by cycling between an inactive GDP bound form and an active GTP bound form In its active GTP bound state Ras binds specifically to the Ras binding domain RBD of Raf 1 to control downstream signaling cascades The most notable members of the Ras subfamily are H Ras N Ras and K Ras mainly for being implicated in many types of cancer Cell Biolabs 96 well Ras Activation ELISA Kit utilizes plate bound Raf 1 RBD to selectively isolate and pull down the active forms of Ras H K and N Ras isoforms from human mouse and rat from purified samples or endogenous lysates Subsequently the captured GTP Ras is detected by an Anti pan Ras Antibody and HRP conjugated secondary antibody Finally following the addition of Chemiluminescent Reagent the reaction is then measured in a plate luminometer Cell Biolabs 96 well Ras Activation ELISA Kit provides a simple and2. fast tool to monitor the activation of Ras Each kit provides sufficient reagents to perform up to 96 assays Related Products 1 STA 400 H H Ras Activation Assay Kit STA 400 K K Ras Activation Assay Kit STA 400 N N Ras Activation Assay Kit STA 401 1 Racl Activation Assay Kit STA 402 Cdc42 Activation Assay Kit STA 403 A RhoA Activation Assay Kit STA 410 Raf 1 RBD Agarose Beads STA 440 96 well Ras Activation ELISA Kit Colorimetric STA 457 Ras Expression Vector Set 10 STA 459 Active Ras Expression Vector Set O ON DAR wWDHD Kit Components Box 1 shipped at room temperature 1 Raf 1 RBD Capture Plate Part No 244101 One solid white 96 well plate 2 5X Assay Lysis Buffer Part No 240102 One 30 mL bottle of 125 mM HEPES pH 7 5 750 mM NaCl 5 NP 40 50 mM MgCl 5 mM EDTA 10 Glycerol 3 Assay Diluent Part No 310804 One 50 mL bottle 4 10X Wash Buffer Part No 310806 One 100 mL bottle 5 Chemiluminescent Reagent A Part No 250102 One 6 mL amber bottle 2 CELL BIOLABS INC P 6 7 Chemiluminescent Reagent B Part No 250103 One 6 mL bottle Plate Sealing Films Part No 244102 One pack of two sealing films Box 2 shipped on blue ice packs 1 Raf 1 RBD 500X Part No 244002 One 40 uL vial 2 Anti pan Ras Antibody 1000X Part No 244003 One 20 uL vial 3 Secondary Antibody HRP Conjugate Part No 244004 One 20 uL vial 4 5 100X GTPyS Part No
3. 0 Proceed to GTPyS GDP Loading for positive and negative controls or the Activation ELISA Assay Protocol Section II Suspension Cells Mi 1 Culture cells and stimulate with Ras activator s as desired 2 Perform a cell count and then pellet the cells by centrifugation 3 4 Completely remove the final PBS wash and add ice cold 1X Assay Lysis Buffer to the cell Aspirate the culture media and wash twice with ice cold PBS pellet 0 5 1 mL per 1 x 10 cells Lyse the cells by repeated pipetting 6 Transfer the lysates to appropriate size tubes and place on ice 7 If nuclear lysis occurs the cell lysates may become very viscous and difficult to pipette If this occurs lysates can be passed through a 27 2 gauge syringe needle 3 4 times to shear the genomic DNA Clear the lysates by centrifugation for 10 minutes 14 000 x g at 4 C 4 A j CELL BIOLABS INC a 9 Collect the supernatant and store samples on ice for immediate use or snap freeze samples and store at 70 C for future use 10 Proceed to GTPyS GDP Loading for positive and negative controls or the Activation ELISA Assay Protocol Section Assay Protocol I 1 GTPyS GDP Loading Positive and Negative Controls Note Samples that will not be GTPyS GDP loaded may be kept on ice during preparation of GTPyS GDP loading samples Aliquot 0 5 mL of each cell lysate to two microcentrifuge tubes Note Typical protein concentrat
4. 240103 One 50 uL vial of 10 mM GTPyS dissolved in sterile water 100X GDP Part No 240104 One 50 uL vial of 100 mM GDP dissolved in sterile water Materials Not Supplied 1 oS a ae ee Stimulated and non stimulated cell or tissue lysates Ras activators Protease inhibitors 0 5 M EDTA in water 1 M MgCl 30 C incubator or water bath Room temperature shaker 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips 10 Multichannel micropipette reservoir 11 96 well Plate Luminometer Storage Upon receipt aliquot and store Raf 1 RBD at 80 C and avoid freeze thaw Aliquot and store the Anti pan Ras Antibody Secondary Antibody GTPyS and GDP components at 20 C and avoid freeze thaw Store all other components at 4 C until their expiration dates Preparation of Reagents 1X Assay Lysis Buffer Mix the 5X stock briefly and dilute to 1X in deionized water Just prior to usage add protease inhibitors such as 1 mM PMSF 10 ug mL leupeptin and 10 ng mL aprotinin 1X Wash Buffer Dilute the 10X Wash Buffer to 1X with deionized water Stir to homogeneity Anti pan Ras Antibody Immediately before use dilute the Anti pan Ras Antibody 1 1000 with Assay Diluent Do not store diluted solutions Secondary Antibody HRP Conjugate Immediately before use dilute the Secondary Antibody HRP Conjugate 1 2500 with Assay Di
5. Protocol Background has been subtracted from data Figure 2 Pan Ras Antibody Specificity Anti pan Ras Antibody specificity to purified H K and N Ras human isoforms by dot blot References 1 Bar Sagi D and Hall A 2000 Cell 103 227 38 2 de Rooij J and Bos J L 1997 Oncogene 14 623 5 7 Jau BIOLABS INC Creating Solutions for Life Science Research Warranty These products are warranted to perform as described in their labeling and in Cell Biolabs literature when used in accordance with their instructions THERE ARE NO WARRANTIES THAT EXTEND BEYOND THIS EXPRESSED WARRANTY AND CELL BIOLABS DISCLAIMS ANY IMPLIED WARRANTY OF MERCHANTABILITY OR WARRANTY OF FITNESS FOR PARTICULAR PURPOSE CELL BIOLABS sole obligation and purchaser s exclusive remedy for breach of this warranty shall be at the option of CELL BIOLABS to repair or replace the products In no event shall CELL BIOLABS be liable for any proximate incidental or consequential damages in connection with the products Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbiolabs com 2013 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing eo CELL BIOLABS INC AN
6. ion of sample is gt 0 5 mg mL Add 10 uL of 0 5 M EDTA to each sample Add 5 uL of 100X GTPYS to one tube positive control and 5 uL of 100X GDP to the other tube negative control Mix and label each tube appropriately 4 Incubate the tubes for 30 minutes at 30 C with agitation au II 1 Stop the loading by adding 33 uL of 1 M MgCl to each tube Mix and place tubes on ice Continue with the Activation ELISA Ras Activation ELISA Note Samples and controls should be thawed maintained on ice just prior to use Step 3 Determine the number of wells to be used and dilute the Raf 1 RBD 1 500 in Assay Diluent Add 100 uL of the diluted Raf 1 RBD to each well of the Raf 1 RBD Capture Plate Incubate at room temperature for 1 hour on an orbital shaker Cover unused wells with a plate sealing film Note Do not store diluted solutions Wash microwell strips 3 times with 250 uL 1X Wash Buffer per well with thorough aspiration between each wash After the last wash empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer Add 50 uL of Ras lysate sample 10 100 ug control or buffer blank per well Each sample should be assayed in duplicate Any sample dilutions should be performed in cold 1X Assay Lysis Buffer Immediately add 50 uL of Assay Diluent to each well 100 uL total volume Incubate at room temperature for 1 hour on an orbital shaker CELL BIOLABS INC a e
7. luent Do not store diluted solutions CELL BIOLABS INC yA o gt Chemiluminescent Reagent Immediately before use mix equal volumes of Chemiluminescent Reagent A with Chemiluminescent Reagent B Do not store diluted solutions Preparation of Samples Note It is advisable to use fresh cell or tissue lysates because GTP Ras is quickly hydrolyzed to GDP Ras frozen lysates stored at 70 C may be used Performing steps at 4 C or on ice may reduce hydrolysis Avoid multiple freeze thaw cycles of lysates I Adherent Cells 1 Culture cells to approximately 80 90 confluence Stimulate cells with Ras activator s as desired 2 Aspirate the culture media and wash twice with ice cold PBS aa a ek Mi Completely remove the final PBS wash and add ice cold 1X Assay Lysis Buffer to the cells 0 5 1 mL per 100 mm tissue culture plate Place the culture plates on ice for 10 20 minutes Detach the cells from the plates by scraping with a cell scraper Transfer the lysates to appropriate size tubes and place on ice If nuclear lysis occurs the cell lysates may become very viscous and difficult to pipette If this occurs lysates can be passed through a 27 2 gauge syringe needle 3 4 times to shear the genomic DNA 8 Clear the lysates by centrifugation for 10 minutes 14 000 x g at 4 C Collect the supernatant and store samples on ice for immediate use or snap freeze samples and store at 70 C for future use 1
8. o 5 Wash microwell strips 5 times with 250 uL 1X Wash Buffer per well with thorough aspiration between each wash After the last wash empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer 6 Add 100 uL of the diluted Anti pan Ras Antibody to each well Incubate at room temperature for 1 hour on an orbital shaker 7 Wash the strip wells 5 times according to step 5 above 8 Add 100 uL of the diluted Secondary Antibody HRP Conjugate to each well Incubate at room temperature for 1 hour on an orbital shaker 9 Wash the strip wells 5 times according to step 5 above Proceed immediately to the next step 10 Add 100 uL of Chemiluminescent Reagent see Preparation of Reagents Section to each well including the blank wells Incubate at room temperature for 5 minutes on an orbital shaker 11 Read the luminescence of each microwell on a plate luminometer e CELL BIOLABS INC i Example of Results The following figure demonstrates typical results seen with Cell Biolabs 96 well Ras Activation ELISA Kit One should use the data below for reference only 20000 i m HeLa Unstimulated Po fl 17500 m HeLa EGF 12500 10000 l x 7500 5000 2500 0 1 5625 3 125 6 25 12 5 25 50 Lysate yg well Figure 1 EGF Stimulation HeLa cells were serum starved for 18 hours before EGF stimulation 50 ng mL for 2 minutes Lysates were then prepared according to Assay
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