RhoC Activation Assay Kit, Trial Size
Contents
1. Figure 1 Rhotekin RBD Agarose beads in color are easy to visualize minimizing potential loss during washes and aspirations CELL BIOLABS INC A AU Assay Principle Lysate 1 Lysate 2 low GTP bound Rho content high GTP bound Rho content o GDP bound Rho GTP bound Rho Rhotekin RBD Agarose Rhotekin RBD Agarose is added mixing and centifugation pull down of GTP bound Rho washing and immunoblotting Related Products STA 400 Pan Ras Activation Assay Kit 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 STA 401 2 Rac2 Activation Assay STA 403 A RhoA Activation Assay STA 403 B RhoB Activation Assay STA 405 RhoA Rac1 Cdc42 Activation Assay Combo Kit 3 oe N oe ee n D CELL BIOLABS INC Pi 10 11 12 STA 410 Rafl RBD Agarose Beads STA 457 Ras Expression Vector Set STA 459 Active Ras Expression Vector Set Kit Components 1 Rhotekin RBD Agarose Part No 240301 T One 200 uL vial of 50 slurry 100 ug Rhotekin RBD in PBS containing 50 glycerol Note Agarose bead appears pink in color for easy identification washing and aspiration 100X GTPyS Part No 240103 T One 20 uL of 10 mM GTPyS dissolved in sterile water 100X GDP Part No 240104 T One 20 uL of 100 mM GDP dissolved in sterile water 5X Assay Lysis Buffer Part No 24012. Product Manual RhoC Activation Assay Kit Trial Size Catalog Number STA 403 C T 5 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 RhoC a 21 kDa protein regulates a variety of biological response pathways that include cell growth cell transformation and tumor invasion Like other small GTPases RhoC regulates molecular events by cycling between an inactive GDP bound form and an active GTP bound form In its active GTP bound state RhoC binds specifically to the Rho binding domain RBD of Rhotekin to control downstream signaling cascades Cell Biolabs RhoC Activation Assay Kit utilizes Rhotekin RBD Agarose beads to selectively isolate and pull down the active form of Rho from purified samples or endogenous lysates Subsequently the precipitated GTP Rho is detected by western blot analysis using an anti RhoC specific monoclonal antibody Assay Principle Cell Biolabs RhoC Activation Assay Kit provides a simple and fast tool to monitor the activation of RhoC The kit includes easily identifiable Rhotekin RBD Agarose beads see Figure 1 pink in color and a RhoC Immunoblot Positive Control for quick RhoC identification This Trial Size kit provides sufficient quantities to perform 5 assays
3. be performed in a plastic bag 3 Wash the blotted membrane three times with TBST 5 minutes each time Incubate the membrane with a secondary antibody e g Goat Anti Mouse IgG HRP conjugate freshly diluted in 5 non fat dry milk TBST for 1 hr at room temperature with constant agitation Wash the blotted membrane three times with TBST 5 minutes each time Use the detection method of your choice We recommend enhanced chemiluminescence reagents from Pierce eo CELL BIOLABS INC yA Example of Results The following figure demonstrates typical results seen with Cell Biolabs RhoC Activation Assay Kit One should use the data below for reference only 28kDa RhoA 17kDa 2 RhoB 14kDa RhoC Figure 2 RhoC Activation Assay Left Image RhoC Immunoblot Positive Control Right Image Demonstrates Anti RhoC monoclonal antibody specificity by dot blot References 1 Ren X D and Schwartz M A 2000 Methods Enzymol 325 264 72 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
4. 02 T Four 2 mL vials of 125 mM HEPES pH 7 5 750 mM NaCl 5 NP 40 50 mM MgCh 5 mM EDTA 10 Glycerol Anti RhoC Mouse Monoclonal Part No 240312 T One 10 uL in PBS pH 7 4 0 05 NaN3 0 1 BSA Note This monoclonal antibody specifically reacts with human mouse and rat RhoC RhoC Immunoblot Positive Control Part No 240311 One 100 uL of partially purified recombinant RhoC from E coli provided ready to use in 1X reducing SDS PAGE Sample Buffer pre boiled Materials Not Supplied O 0 ee a ee eS ee gt Stimulated and non stimulated cell lysates RhoC activators Protease inhibitors 0 5 M EDTA in water 1 M MgCl 30 C incubator or water bath 4 C tube rocker or shaker 2X reducing SDS PAGE sample buffer Electrophoresis and immunoblotting systems Immunoblotting wash buffer such as TBST 10 mM Tris HCl pH 7 4 0 15 M NaCl 0 05 Tween 20 Immunoblotting blocking buffer TBST containing 5 Non fat Dry Milk 12 13 14 PVDF or nitrocellulose membrane Secondary Antibody ECL Detection Reagents CELL BIOLABS INC P Storage Store all kit components at 20 C The 5X Assay Lysis Buffer may be stored at either 20 C or 4 C Avoid multiple freeze thaw cycles Preparation of Reagents e 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 pg mL leupeptin and 10 ug mL aprotinin Prepar
5. ation of Samples Note It is advisable to use fresh cell lysates because GTP RhoC is quickly hydrolyzed to GDP RhoC 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 RhoC activator s as desired 2 Aspirate the culture media and wash twice with ice cold PBS o 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 ae oe a 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 9 Collect the supernatant and store samples on ice for immediate use or snap freeze and store at 70 C for future use 10 Proceed to GTPyS GDP Loading for positive and negative controls or Pull Down Assay II Suspension Cells 1 Culture cells and stimulate with RhoC activator s as desired 2 Perform a cell count and then pellet the cells by centrifugation 3 Aspirate the cultur
6. e media and wash twice with ice cold PBS 4 Completely remove the final PBS wash and add ice cold 1X Assay Lysis Buffer to the cell pellet 0 5 1 mL per 1 x 10 cells a Lyse the cells by repeated pipetting 6 Transfer the lysates to appropriate size tubes and place on ice 5 CELL BIOLABS INC 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 and store at 70 C for future use 10 Proceed to GTPyS GDP Loading for positive and negative controls or Pull Down Assay Assay Protocol Important Note Before running any Small GTPase pulldown assay it is always a good practice to run a Western Blot directly on the cell lysate using the antibody provided in this kit For example load 5 ug 10 ug and 20 ug of lysate onto an SDS PAGE gel transfer and blot When proceeding with the pulldown assay use 100 times the amount of lysate that gave you a clear band of your desired small GTPase in the direct Western blot For example if the 5 ug band was faint but the 10 ug band was clear and strong use 100 x 10 ug 1 mg of lysate in the assay Using sufficient lysate in the pulldown assay is critical to success I GTP
7. 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 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing oa CELL BIOLABS INC yA AU
8. ting each time 9 After the last wash pellet the beads and carefully remove all the supernatant 10 Resuspend the bead pellet in 40 uL of 2X reducing SDS PAGE sample buffer 11 Boil each sample for 5 minutes 12 Centrifuge each sample for 10 seconds at 14 000 x g III Electrophoresis and Transfer 1 Load 20 uL well of pull down supernatant to a polyacrylamide gel Also it s recommended to include a pre stained MW standard as an indicator of a successful transfer in step 3 Note If desired 10 uL well of RhoC Immunoblot Positive Control provided ready to use pre boiled can be added as an immunoblot positive control Perform SDS PAGE as per the manufacturer s instructions Transfer the gel proteins to a PVDF or nitrocellulose membrane as per the manufacturer s instructions IV Immunoblotting and Detection all steps are at room temperature with agitation 1 Following the electroblotting step immerse the PVDF membrane in 100 Methanol for 15 seconds and then allow it to dry at room temperature for 5 minutes Note If Nitrocellulose is used instead of PVDF this step should be skipped Block the membrane with 5 non fat dry milk in TBST for 1 hr at room temperature with constant agitation Incubate the membrane with Anti RhoC Antibody freshly diluted 1 200 to 1 1000 in 5 non fat dry milk TBST for 1 2 hr at room temperature with constant agitation Note To conserve antibody incubations should
9. yS GDP Loading Positive and Negative Controls Note Samples that will not be GTPyS GDP loaded may be kept on ice during the loading of controls 1 Aliquot 0 5 1 mL of each cell lysate to two microcentrifuge tubes Note Typical protein content sample is gt 0 5 mg Adjust the volume of each sample to 1 mL with 1X Assay Lysis Buffer 3 Add 20 uL of 0 5 M EDTA to each sample Add 10 uL of 100X GTPYS to one tube positive control and 10 uL of 100X GDP to the other tube negative control Mix and label each tube appropriately Incubate the tubes for 30 minutes at 30 C with agitation Stop the loading by adding 65 uL of 1 M MgCl to each tube Mix and place tubes on ice Continue with Pull Down assay II RhoC Pull Down Assay 1 Sh Oy Se oe a Aliquot 0 5 1 mL of cell lysate treated with RhoC activators or untreated to a microcentrifuge tube Adjust the volume of each sample to 1 mL with 1X Assay Lysis Buffer Thoroughly resuspend the Rhotekin RBD Agarose bead slurry by vortexing or titurating Quickly add 40 uL of resuspended bead slurry to each tube including GTPyS GDP controls Incubate the tubes at 4 C for 1 hour with gentle agitation Pellet the beads by centrifugation for 10 seconds at 14 000 x g Aspirate and discard the supernatant making sure not to disturb remove the bead pellet 6 eo CELL BIOLABS INC a 8 Wash the bead 3 times with 0 5 mL of 1X Assay Buffer centrifuging and aspira
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