Rac2 Activation Assay Kit
Contents
1. Product Manual Rac2 Activation Assay Kit Catalog Number STA 401 2 20 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 Rac2 a 21 kDa protein belongs to the family of Rho GTPases regulates a variety of biological response pathways that include cell motility cell division gene transcription and cell transformation Like other small GTPases Rac2 regulates molecular events by cycling between an inactive GDP bound form and an active GTP bound form In its active GTP bound state Rac2 binds specifically to the p21 binding domain PBD of p21 activated protein kinase PAK to control downstream signaling cascades Cell Biolabs Rac2 Activation Assay Kit utilizes PAK PBD Agarose beads to selectively isolate and pull down the active form of Rac from purified samples or endogenous lysates Subsequently the precipitated GTP Rac is detected by western blot analysis using an anti Rac2 specific polyclonal antibody see Assay Principle Cell Biolabs Rac2 Activation Assay Kit provides a simple and fast tool to monitor the activation of Rac2 The kit includes easily identifiable PAK1 PBD Agarose beads see Figure 1 pink in color and a Rac2 Immunoblot Positive Control for quick Rac2 identification Each ki2. 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 ug mL aprotinin Preparation of Samples Note It is advisable to use fresh cell lysates because GTP Rac2 is quickly hydrolyzed to GDP Rac2 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 Rac2 activator s as desired 2 Aspirate the culture media and wash twice with ice cold PBS m 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 QUO Io 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 C
3. 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 GTPyS GDP Loading Positive and Negative Controls Note Samples that will not be GIPyS GDP loaded may be kept on ice during the loading of controls l Aliquot 0 5 1 mL of each cell lysate to two microcentrifuge tubes Note Typical protein content sample is 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 Rac2 Pull Down Assay l we M Se OM ge Aliquot 0 5 1 mL of cell lysate treated with Rac2 activators or untreated to a microcentrifuge tube Adjust the volume of each sample to 1 mL with 1X Assay Lysis Buffer Thoroughly resuspend the PAK PBD 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 6 CELL BIOLABS INC e 7 Aspirate and discard the superna
4. non fat dry milk TBST for 1 2 hr at room temperature with constant agitation Note To conserve antibody incubations should 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 Rabbit IgG HRP conjugate freshly diluted in 5 non fat dry milk TBST for 1 hr at room temperature with constant agitation 5 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 e CELL BIOLABS INC P AU Example of Results The following figure demonstrates typical results seen with Cell Biolabs Rac2 Activation Assay Kit One should use the data below for reference only Racl Rac2 Figure 2 Rac2 Activation Assay Left Image Rac2 Immunoblot Positive Control Right Image Demonstrates Anti Rac2 polyclonal antibody specificity by dot blot References 1 Raftopoulou M and Hall A 2004 Dev Biol 265 23 32 2 Bar Sagi D and Hall A 2000 Cell 103 227 38 3 Benard V Bohl B P and Bokoch G M 1999 J Biol Chem 274 13198 13204 Recent Product Citation Baetta R et al 2015 Atorvastatin reduces long pentraxin 3 expression in vascular cells by inhibiting protein geranylgeranylation Vascul Pharmacol doi 10 1016 j vph 2014 11 008 Warranty These products are warranted to pe
5. olved in sterile water 5X Assay Lysis Buffer Part No 240102 One bottle 30 mL of 125 mM HEPES pH 7 5 750 mM NaCl 5 NP 40 50 mM MgCl 5 mM EDTA 10 Glycerol Anti Rac2 Rabbit Polyclonal Part No 240112 One vial 40 uL in PBS pH 7 4 0 05 NaN3 0 1 BSA Note This polyclonal antibody specifically reacts with human and mouse Rac2 Additional unknown higher MW proteins may be detected in some preparations Rac2 Immunoblot Positive Control Part No 240111 One vial 100 uL of partially purified recombinant Rac2 from E coli provided ready to use in 1X reducing SDS PAGE Sample Buffer pre boiled Materials Not Supplied SOC e Oy OE Ye Ir e 11 12 13 14 Stimulated and non stimulated cell lysates Rac2 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 PVDF or nitrocellulose membrane Secondary Antibody ECL Detection Reagents CELL BIOLABS INC ae 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
6. rform 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 N CELL BIOLABS INC eue Contact Information Cell Biolabs Inc 7158 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech G9 cellbiolabs com www cellbiolabs com 2010 2015 Cell Biolabs Inc All rights reserved No part of these works may be reproduced in any form without permissions in writing e CELL BIOLABS INC AN
7. t provides sufficient quantities to perform 20 assays Figure 1 PAK RBD Agarose beads in color are easy to visualize minimizing potential loss during washes and aspirations CELL BIOLABS INC M AU Assay Principle Lysate 1 Lysate 2 low GTP bound Rac content high GTP bound Rac content i m Kad GDP bound Rac 9 GTP bound Rac Pak1 PBD Agarose Y PAK1 PBD Agarose Y is added A aaa a mixing and centifugation pull down of GTP bound Rac 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 402 Cdc42 Activation Assay Kit STA 403 A RhoA Activation Assay Kit STA 405 RhoA Rac1 Cdc42 Activation Assay Combo Kit STA 410 Rafl RBD Agarose Beads i e dL Qe u e UO po A CELL BIOLABS INC _ ee ee 10 11 STA 457 Ras Expression Vector Set STA 459 Active Ras Expression Vector Set Kit Components 1 PAKI PBD Agarose Part No STA 411 One vial 800 uL of 50 slurry 400 ug PBD in PBS containing 50 glycerol Note Agarose bead appears pink in color for easy identification washing and aspiration 100X GTPyS Part No 240103 One vial 50 uL of 10 mM GTPYS dissolved in sterile water 100X GDP Part No 240104 One vial 50 uL of 100 mM GDP diss
8. tant making sure not to disturb remove the bead pellet 8 Wash the bead 3 times with 0 5 mL of 1X Assay Buffer centrifuging and aspirating 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 l 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 Rac2 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 3 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 Rac2 Antibody freshly diluted 1 1000 in 5
9. ulture cells and stimulate with Rac2 activator s as desired 2 Perform a cell count and then pellet the cells by centrifugation 3 Aspirate the culture 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 5 Lyse the cells by repeated pipetting CELL BIOLABS INC 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 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
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