EpiQuik™ In Vivo Protein Sumoylation Assay Ultra Kit
Contents
1. EpiQuik In Vivo Protein Sumoylation Assay Ultra Kit Base Catalog P 8003 PLEASE READ THIS ENTIRE USER GUIDE BEFORE USE Uses The EpiQuik n Vivo Protein Sumoylation Assay Ultra Kit is very suitable for measuring in vivo protein sumoylation from multiple mammalian cells tissues including human mouse and rat Nuclear extracts can be prepared by using your own successful method For your convenience and the best results Epigentek offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear extracts can be used immediately or stored at 80 C for future use Input Material Input material is nuclear extracts The amount of nuclear extracts for each assay can be 1 ug 20 ug with an optimal range of 5 to 10 yg Internal Control The positive control SUMO protein and negative control non immune IgG are provided in this kit for the quantification of sumoylation Because percentage of sumoylation can vary from tissue to tissue and from normal and diseased states it is advised to run replicate samples to ensure that the signal generated is validated Precautions To avoid cross contamination carefully pipette the sample or solution into the strip wells Use aerosol barrier pipette tips and always change pipette tips between liquid transfers Wear gloves throughout the entire procedure In case of contact between gloves and sample change gloves immediately 110 Bi County Blvd Ste 122 Farmingdale NY2. 11735 Page 1 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2013 12 18 Epigentek Group Inc All rights reserved Products are for research use only P 8003 CONTENTS Component 48 Assays Cat P 8003 48 96 Assays Cat P 8003 96 Storage Upon Receipt WB 10X Wash Buffer 15 ml 30 ml 4 C BS Binding Solution 10 ml 20 ml 47 NC Negative Control 250 g ml 10 pl 20 ul 4 BB Blocking Buffer 10 ml 20 ml 47 SAB SUMO Assay Buffer 5 ml 10 ml 4 PC Positive Control 1 ug l 10 ul 20 ul 20 C DA Detection Antibody 1000X 5 ul 10 pl 20 C CD Color Developer 5 ml 10 ml 4 SS Stop Solution 5 ml 10 ml RT 8 Well Assay Sirips With Frame 6 12 4 User Guide 1 1 RT Spin the solution down to the bottom prior to use SHIPPING amp STORAGE The kit is shipped in two parts the first part at ambient room temperature and the second part on frozen ice packs at 4 C Upon receipt 1 Store PC and DA at 20 C away from light 2 Store WB BS NC BB SAB CD and 8 Well Assay Strips at 4 C away from light and 3 Store remaining components at room temperature away from light All components of the kit are stable for 6 months from the date of shipment when stored properly Note 1 Check if WB 10X Wash Buffer contains salt precipitates before use If so warm at room temperature or 37 C and sh
3. blue The color will change to yellow after adding SS and the absorbance should be read on a microplate reader within 2 to 10 min at 450 nm with an optional reference wavelength of 655 nm Note 1 Most microplate readers have the capability to carry out dual wavelength analysis and will automatically subtract reference wavelength absorbance from the test wavelength absorbance If your plate reader does not have this capability the plate can be read twice once at 450 nm and once at 655 nm Then manually subtract the 655 nm ODs from 450 nm ODs 2 if the strip well microplate frame does not fit in the microplate reader transfer the solution to a standard 96 well microplate 6 Sumoylation Calculation Calculate sumoylation of the targeted proteins For simple calculation 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 7 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 18 Epigentek Group Inc All rights reserved Products are for research use only P 8003 io co on A se WE OD treated sample negative control Sumoylation x 100 OD untreated sample negative control For accurate calculation 1 Plot Delta OD values positive control OD Blank OD versus amount of PC added in the wells and determine the slope as delta OD ng 2 Calculate intensity of the conjugated SUMO using the following formula OD sample negative c
4. the Blocking Buffer from each well Wash each well two times with 150 ul of the Diluted WB each time 3 Sumoylated Protein Capture a b Blank Wells Add 50 ul of SAB SUMO Assay Buffer Standard Control Wells Add 50 ul of SAB to each standard well Sample Wells Add 50 ul of SAB and then add 5 10 ug of nuclear proteins total nuclear protein volume should be no more than 10 ul Negative Control Well Add 50 ul of SAB to each negative control well Tightly cover strip wells with Parafilm M to avoid evaporation and incubate at 37 C for 60 min Remove the reaction solution from each well Wash each well two times with 150 ul of the Diluted WB each time 4 Detection Antibody Binding a Add 50 ul of the Diluted DA to each well then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 min Remove the Diluted DA from each well Wash each well four times with 150 ul of the Diluted WB each time Note Ensure any residual wash buffer in the wells is removed as much as possible at each wash step 5 Signal Detection a Add 100 ul of CD Color Developer to each well and incubate at room temperature for 1 to 10 min away from light Begin monitoring color change in the sample wells and control wells The CD solution will turn blue in the presence of sufficient SUMO products Add 100 ul of SS Stop Solution to each well to stop enzyme reaction when color in the positive control wells turns medium
5. well is not washed sufficiently Check if the wash at each step is performed according to the protocol Overdevelopment Decrease development time in Step 4a RELATED PRODUCTS Nuclear Extract Preparation OP 0002 Sumoylation Assay EpiQuik n Vivo HDAC1 Sumoylation Assay Kit P 8002 EpiQuik Nuclear Extraction Kit Sumoylation Antibodies A 8144 A 8143 SUMO2 Monoclonal Antibody 2C7 1A11 SUMO3 Monoclonal Antibody 4G11 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 9 Printed 2013 12 18 P 8003 Sumoylation Proteins E28000 E28001 E28002 E28003 E28004 E28005 SUMO 1 Protein SUMO 1 biotin Protein SUMO 2 Protein SUMO 2 biotin Protein SUMO 3 Protein SUMO 3 biotin Protein 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 10 Printed 2013 12 18 P 8003
6. Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2013 12 18 TROUBLESHOOTING Problem Possible Causes Suggestions No signal for the sample Antibodies are not properly coated Ensure the concentration of the antibody solution Antibodies are not IP grade Ensure the antibodies can be used for IP The protein sample is not properly extracted Ensure the protein extraction protocol is for nuclear protein extraction The protein amount is added into well insufficiently Ensure extract contains a sufficient amount of protein Nuclear extracts are stored incorrectly Ensure the nuclear extracts are stored at 80 C Reagents are added incorrectly Check if reagents are added in order and if some steps of the procedure were omitted by mistake Incubation time and temperature are incorrect Ensure the incubation time and temperature described in the protocol are followed correctly Absence of sumoylation N A High background present for the negative control background The negative control wells are contaminated with antibodies Ensure only negative control is added The wells are not sufficiently blocked with BB Increase blocking time to 60 90 minutes The
7. ake the buffer until the salts are re dissolved and 2 check if a blue color is present in CD Color Developer which would indicate a contamination of the solution and should not be used To avoid contamination transfer the amount of CD required into a secondary container tube or vial before adding CD into the assay wells MATERIALS REQUIRED BUT NOT SUPPLIED oO Oo 0 Ama oO Adjustable pipette or multiple channel pipette Multiple channel pipette reservoirs Aerosol resistant pipette tips Microplate reader capable of reading absorbance at 450 nm 1 5 ml microcentrifuge tubes Incubator for 37 C incubation 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Page 2 Printed 2013 12 18 P 8003 O Antibodies against proteins of interest O Nuclear protein samples O Parafilm M GENERAL PRODUCT INFORMATION Quality Control Each lot of the EpiQuik n Vivo Protein Sumoylation Assay Ultra Kit is tested against predetermined specifications to ensure consistent product quality Epigentek guarantees the performance of all products in the manner described in our product instructions Product Warranty If this product does not meet your expectations simply contact our technical support unit or your regional distributor We also encourage you to con
8. g solution and read absorbance Fig 1 Schematic Procedure for Using the EpiQuik n Vivo Protein Sumoylation Assay Ultra Kit 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 4 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Printed 2013 12 18 Epigentek Group Inc All rights reserved Products are for research use only P 8003 0 8 0 6 OD 450 nr 0 4 0 2 0 100 200 300 400 500 SUMO protein ng Fig 2 Illustrated standard curve generated with sumo protein control PROTOCOL For the best results please read the protocol in its entirety prior to starting your experiment Starting Materials Input Amount The amount of nuclear extracts for each assay can be between 1 and 20 pg with an optimal range of 5 to 10 ug Nuclear Extraction You can use your method of choice for preparing nuclear extracts from the treated and untreated samples Epigentek also offers a nuclear extraction kit Cat OP 0002 optimized for use with this kit Nuclear extracts should be stored in aliquots at 80 C until use 1 Working Buffer and Solution Preparation Prepare Diluted WB 48 Assay Kit Add 13 ml of WB to 117 ml of distilled water and adjust pH to 7 2 7 5 96 Assay Kit Add 26 ml of WB to 234 ml of distilled water and adjust pH to 7 2 7 5 This Diluted WB can now be stored at 4 C for up to six months Prepare Diluted DA Detection Antibody Solution Dilute DA w
9. he number of strip wells required for your experiment It is advised to run replicate samples include blank and positive controls to ensure that the signal generated is validated Carefully remove unneeded strip wells from the plate frame and place them back in the bag seal the bag tightly and store at 4 C b Blank Wells Add 100 ul of BS to each blank well c Standard Control Wells Add 98 ul of BS to each well and then add 2 ul of Diluted PC to each standard control well with a minimum of six wells each at a different concentration between 10 and 500 ng well based on the dilution chart in Step 1d see Table 1 under the Suggested Strip Well Setup section as an example d Sample Wells Add 100 ul of the diluted antibody solution against the protein of interest to each sample well from Step 1c e Negative Control Well Add 98 ul of BS to each well and then add 2 ul 0 5 ug of NC Negative Control f Tightly cover strip well microplate with Parafilm M to avoid evaporation and incubate at 37 C for 90 to 120 min g Remove the solutions from each well Add 150 ul of BB Blocking Buffer to the wells and incubate at room temperature for 45 minutes 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 6 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com m Web www epigentek com Epigentek Group Inc All rights reserved Products are for research use only Printed 2013 12 18 P 8003 h Remove
10. ith Diluted WB at a ratio of 1 1000 i e add 1 ul of DA to 1000 ul of Diluted WB 50 ul of Diluted DA will be required for each assay well Prepare diluted antibodies that are specific for the protein of interest The antibodies should be IP grade 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 5 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2013 12 18 Epigentek Group Inc All rights reserved Products are for research use only P 8003 Dilute the antibodies to 2 ug ml with BS Binding Solution 100 ul of diluted antibodies are required for each sample well Prepare Diluted Positive Control for Standard Curve Suggested Standard Curve Preparation First dilute PC Positive Control with BS to 250 ng ul by adding 5 ul of PC to 15 ul of BS Then further prepare five concentrations by combining the 250 ng ul Diluted PC with BS into final concentrations of 5 10 25 50 100 and 250 ng ul according to the following dilution chart puce a Ee ip e P 1 1 0 ul 49 0 ul 5 ng ul 2 1 0 ul 24 0 ul 10 ng ul 3 1 0 ul 9 0 ul 25 ng ul 4 1 0 ul 4 0 ul 50 ng ul 5 2 0 ul 3 0 ul 100 ng ul 6 5 0 ul 0 0 ul 250 ng ul Note Keep each of the diluted solutions except Diluted WB on ice until use Any remaining diluted solutions other than Diluted WB should be discarded if not used within the same day 2 Antibody and Control Coating a Predetermine t
11. need for affinity chromatography and Western blotting e Flexible choice of antibody of interest allows the detection of sumoylation of multiple target proteins simultaneously e Use of optimized detection antibody eliminates the step for detection solution preparation increasing detection sensitivity and assay convenience e The positive control SUMO protein and negative control un sumoylated non immune IgG protein allows protein sumoylation to be quantified e Strip microplate format makes the assay flexible manual or high throughput analysis e Reliable and consistent assay conditions PRINCIPLE amp PROCEDURE The EpiQuik n Vivo Protein Sumoylation Assay Ultra Kit is designed for measuring sumoylation of the targeted proteins Sumoylation of the targeted proteins is indicated by SUMO conjugated to these proteins In an assay with this kit the antibodies specific to the targeted proteins are stably bound to the strip wells and the targeted proteins are captured by these antibodies Sumoylation of the targeted proteins are detected by recognition of SUMO conjugated to these proteins with an anti SUMO antibody The ratio or intensity of the sumoylation which is proportional to the conjugated SUMO amount can be quantified through the signal report color development system Nuclear extracts y Incubate with target protein antibody coated on the wells for 60 min v Add SUMO detection antibody y Add color developin
12. ontrol Sumoylation intensity x 1000 ng mg protein slope x protein amount added ug SUGGESTED STRIP WELL SETUP Table 1 Standard Curve Preparation The suggested strip well plate setup for standard curve preparation in a 48 assay format for a 96 assay format Strips 7 to 12 can be configured as Sample The controls and samples can be measured in duplicate Well Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B PC 10 ng PC 10 ng Sample Sample Sample Sample c PC 20 ng PC 20 ng Sample Sample Sample Sample D PC 50 ng PC 50 ng Sample Sample Sample Sample E PC 100 ng PC 100 ng Sample Sample Sample Sample F PC 200 ng PC 200 ng Sample Sample Sample Sample G PC 500 ng PC 500 ng Sample Sample Sample Sample H NC NC Sample Sample Sample Sample Table 2 Approximate amount of required buffers and solutions for defined assay wells based on the protocol Reagents 1 well 1 strip 2 strips 6 strips 12 strips 8 wells 16 wells 48 wells 96 wells Diluted WB 2 5 ml 20 ml 40 ml 120 ml 240 ml BS 120 ul 1000 ul 2000 ul 6000 ul 12000 ul NC N A 2 ul 4ul 4 ul 8 ul BB 150 ul 1200 ul 2400 ul 7200 ul 14 400 ul SAB 50 ul 400 ul 800 ul 2400 ul 4800 ul PC N A N A 3 ul optional 5 ul 10 ul Diluted DA 50 ul 400 ul 800 ul 2400 ul 4800 ul CD 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml SS 0 1 ml 0 8 ml 1 6 ml 4 8 ml 9 6 ml 110
13. tact us if you have any suggestions about product performance or new applications and techniques Safety Suitable lab coat disposable gloves and proper eye protection are required when working with this product Product Updates Epigentek reserves the right to change or modify any product to enhance its performance and design The information in this User Guide is subject to change at any time without notice Thus only use the User Guide that was supplied with the kit when using that kit Usage Limitation The EpiQuik n Vivo Protein Sumoylation Assay Ultra Kit is for research use only and is not intended for diagnostic or therapeutic application Intellectual Property The EpiQuik n Vivo Protein Sumoylation Assay Ultra Kit and methods of use contain proprietary technologies by Epigentek EpiQuik is a trademark of Epigentek Group Inc A BRIEF OVERVIEW Sumoylation is a post translational modification involved in various cellular processes such as nuclear cytosolic transport transcriptional regulation apoptosis protein stability response to stress and progression through the cell cycle SUMO proteins are similar to ubiquitin There are 3 confirmed SUMO isoforms in humans SUMO 1 SUMO 2 and SUMO 3 SUMO 2 3 show a high degree of similarity to each other and are distinct from SUMO 1 Sumoylation is directed by an enzymatic cascade analogous to that involved in ubiquitination Sumoylation of target proteins in vivo has been sho
14. wn to cause a number of different outcomes including altered localization and binding partners In many cases sumoylation of transcriptional regulators correlates with inhibition of transcription Most sumoylated proteins contain the tetrapeptide consensus motif Y K x D E where is a hydrophobic residue K is the lysine conjugated to SUMO x is any amino acid aa and D or E is an acidic residue Thus detection of in vivo protein sumoylation SUMO conjugation would provide useful information for understanding SUMO modification that emerges as an important control mechanism regulating the activity of many nuclear proteins There are few methods currently available for measuring in vivo protein sumoylation The EpiQuik In Vivo Universal Protein Sumoylation Assay Kit addresses this problem and uses a proprietary and unique procedure to measure in vivo protein sumoylation Epigentek continues to innovate with the 110 Bi County Blvd Ste 122 Farmingdale NY 11735 Page 3 Tel 1 877 374 4368 m Fax 1 718 484 3956 m E mail info epigentek com Web www epigentek com Printed 2013 12 18 Epigentek Group Inc All rights reserved Products are for research use only P 8003 development of EpiQuik n Vivo Protein Sumoylation Assay Ultra Kit which allows increased detection sensitivity and assay convenience The ultra kit has the following features e Fast procedure which can be finished within 5 hours e Direct colorimetric assay without the
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