ab Protein Sumoylation Assay Ultra Kit

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1 ab Protein Sumoylation Assay Ultra Kit Instructions for Use For the measuring in vivo protein sumoylation in various samples This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 1 September 2014

2 Table of Contents INTRODUCTION 1. BACKGROUND 2 2. ASSAY SUMMARY 4 GENERAL INFORMATION 3. PRECAUTIONS 5 4. STORAGE AND STABILITY 5 5. MATERIALS SUPPLIED 6 6. MATERIALS REQUIRED, NOT SUPPLIED 6 7. LIMITATIONS 7 8. TECHNICAL HINTS 7 ASSAY PREPARATION 9. REAGENT PREPARATION STANDARD PREPARATION SAMPLE PREPARATION PLATE PREPARATION 10 ASSAY PROCEDURE 13. ASSAY PROCEDURE 11 DATA ANALYSIS 14. ANALYSIS 14 RESOURCES 15. TROUBLESHOOTING NOTES 18 Discover more at 1

3 INTRODUCTION 1. BACKGROUND 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 shown 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 Ψ-K-x-D/E where Ψ 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. Abcam s Protein Sumoylation Assay Ultra Kit (ab185915) addresses this problem and uses a proprietary and unique procedure to measure in vivo protein sumoylation, allowing increased detection sensitivity and assay convenience. The Ultra kit has the following features: Fast procedure, which can be finished within 5 hours. Direct colorimetric assay without the need for affinity chromatography and Western blotting Flexible choice of antibody of interest allows the detection of sumoylation of multiple target proteins simultaneously Use of optimized detection antibody eliminates the step for detection solution preparation, increasing detection sensitivity and assay convenience Discover more at 2

4 INTRODUCTION The positive control (SUMO protein) and negative control (unsumoylated non-immune IgG protein) allows protein sumoylation to be quantified Strip microplate format makes the assay flexible: manual or high throughput analysis Reliable and consistent assay conditions Abcam s Protein Sumoylation Assay Ultra Kit (ab185915) 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. Nuclear extracts can be used immediately or stored at 80 C for future use. This 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. Discover more at 3

5 INTRODUCTION 2. ASSAY SUMMARY Start with nuclear extracts Incubate with target protein antibody coated on the wells Add SUMO detection antibody Add color developing solution and then measure absorbance Discover more at 4

6 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit as given in the table upon receipt and protect from light. Observe the storage conditions for individual prepared components in sections 9 & 10. For maximum recovery of the products, centrifuge the original vial prior to opening the cap. Check if Wash Buffer contains salt precipitates before use. If so, warm at room temperature or 37 C and shake the buffer until the salts are redissolved. Check if a blue color is present in Color Developer, which would indicate a contamination of the solution and should not be used. To avoid contamination, transfer the amount of Color Developer required into a secondary container (tube or vial) before adding Color Developer into the assay wells. Discover more at 5

7 GENERAL INFORMATION 5. MATERIALS SUPPLIED Item 48 Tests 96 Tests Storage Condition (Before Preparation) 10X Wash Buffer 15 ml 30 ml 4 C Binding Solution 10 ml 20 ml 4 C Negative 10 µl 20 µl 4 C Blocking Buffer 10 ml 20 ml 4 C SUMO Assay Buffer 5 ml 10 ml 4 C 10 µl 20 µl -20 C Detection Antibody 5 µl 10 µl -20 C Color Developer 5 ml 10 ml 4 C Stop Solution 5 ml 10 ml RT 8-Well Assay Strips (With Frame) C 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: 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 Antibodies against proteins of interest Nuclear protein samples Parafilm M or aluminium foil Discover more at 6

8 GENERAL INFORMATION 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures Do not use kit or components if it has exceeded the expiration date on the kit labels Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted Any variation in operator, pipetting technique, washing technique, incubation time or temperature, and kit age can cause variation in binding 8. TECHNICAL HINTS Avoid foaming or bubbles when mixing or reconstituting components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Complete removal of all solutions and buffers during wash steps. This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. Discover more at 7

9 ASSAY PREPARATION 9. REAGENT PREPARATION Prepare fresh reagents immediately prior to use X Wash Buffer Add the volume specified in the table below of 10X Wash Buffer to distilled water and adjust to ph Volume to Dilute (ml) Volume distilled water (ml) Total Volume (ml) 48 Tests Tests The 1X Wash Buffer can now be stored at 4 C for up to six months. 9.2 Detection Antibody Solution Dilute Detection Antibody with Diluted Wash Buffer at a ratio of 1/1000 (i.e. add 1 µl of Detection Antibody to 1000 µl of Diluted Wash Buffer). 50 µl of Diluted Detection Antibody will be required for each assay well. Keep each of diluted solution on ice until use. Any remaining diluted solutions should be discarded if not used within the same day. 9.3 Specific Antibodies Prepare diluted antibodies that are specific for the protein of interest. The antibodies should be IP-grade: Dilute the antibodies to 2 μg/ml with Binding Solution. 100 μl of diluted antibodies are required for each sample well. Keep each of diluted solution on ice until use. Any remaining diluted solutions should be discarded if not used within the same day. Discover more at 8

10 ASSAY PREPARATION 10. STANDARD PREPARATION Suggested Standard Curve Preparation: First, dilute with Binding Solution to 250 ng/μl by adding 5 μl of to 15 μl of Binding Solution. Then, further prepare five concentrations by combining the 250 ng/μl Diluted with Binding Solution into final concentrations of 5, 10, 25, 50, 100, and 250 ng/μl according to the following dilution chart: Tube (µl) Binding Solution (µl) Final Conc (ng/µl) Keep each of diluted solution on ice until use. Any remaining diluted solutions should be discarded if not used within the same day. 11. SAMPLE PREPARATION Input Amount: The amount of nuclear extracts for each assay can be between 1 and 20 μg with an optimal range of 5 to 10 μg. Nuclear Extraction: You can use your method of choice for preparing nuclear extracts. Abcam offers a Nuclear Extraction Kit (ab113474) optimized for use with this kit. Nuclear extract should be stored in aliquots at 80 C until use. Discover more at 9

11 ASSAY PREPARATION 12. PLATE PREPARATION The suggested strip-well plate setup for standard curve preparation in a 48- assay format (in a 96-assay format, Strips 7 to 12 can be configured as Sample). The controls and samples can measured in duplicate. Well # Strip 1 Strip 2 Strip 3 Strip 4 Strip 5 Strip 6 A Blank Blank Sample Sample Sample Sample B C D E F G H 10ng 20ng 50ng 100ng 200ng 500ng Negative 10ng 20ng 50ng 100ng 200ng 500ng Negative Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Discover more at 10

12 ASSAY PROCEDURE 13. ASSAY PROCEDURE Internal : 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 Antibody and Coating Predetermine the number of strip wells required for your experiment. Carefully remove un-needed strip wells from the plate frame and place them back in the bag (seal the bag tightly and store at 4 C) Blank Wells: Add 100 µl of Binding Solution to each blank well Standard Wells: Add 98 µl of Binding Solution to each well and then add 2 µl of Diluted to each standard control well with a minimum of six wells, each at a different concentration between 10 and 500 ng/well (see Standard Preparation Section) Sample Wells: Add 100 µl of the diluted antibody solution against the protein of interest to each sample well (see Standard Preparation Section) Negative Well: Add 98 µl of Binding Solution to each well and then add 2 µl (0.5 µg) of Negative Tightly cover strip-well microplate with Parafilm M to avoid evaporation and incubate at 37 C for 90 to 120 minutes Remove the solutions from each well. Add 150 µl of Blocking Buffer to the wells and incubate at room temperature for 45 minutes Remove the Blocking Buffer from each well. Wash each well two times with 150 µl of the Diluted Wash Buffer each time. Discover more at 11

13 ASSAY PROCEDURE 13.2 Sumoylation Protein Capture Blank Wells: Add 50 µl of SUMO Assay Buffer Standard Wells: Add 50 µl of SUMO Assay Buffer to each standard well Sample Wells: Add 50 µl of SUMO Assay Buffer and then add 5-10 µg of nuclear proteins (total nuclear protein volume should be no more than 10 µl) Negative Well: Add 50 µl of SUMO Assay Buffer to each negative control well Tightly cover strip-wells with Parafilm M to avoid evaporation and incubate at 37 C for 60 minutes Remove the reaction solution from each well. Wash each well two times with 150 µl of the Diluted Wash Buffer each time Detection Antibody Binding Add 50 µl of the Diluted Detection Antibody to each well, then cover with Parafilm M or aluminium foil and incubate at room temperature for 60 minutes Remove the Diluted Detection Antibody from each well Wash each well four times with 150 µl of the Diluted Wash Buffer each time. Note: Ensure any residual wash buffer in the wells is removed as much as possible at each wash step Signal Detection Add 100 µl of Color Developer to each well and incubate at room temperature for 1 to 10 minutes away from light. Begin monitoring color change in the sample wells and control wells. The Color Developer solution will turn blue in the presence of sufficient SUMO products Add 100 µl of Stop Solution to each well to stop enzyme reaction when color in the positive control wells turns medium blue. The color will change to yellow after adding Stop Solution and the absorbance should be read on a microplate reader Discover more at 12

14 ASSAY PROCEDURE within 2 to 10 minutes 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. Discover more at 13

15 DATA ANALYSIS 14. ANALYSIS Sumoylation Calculation Simple Calculation: % Sumoylation = OD (treated sample negative control) OD (untreated sample negative control) 100% For accurate or specific activity calculation: Plot Delta OD values (positive control OD Blank OD) versus amount of added in the wells and determine the slope as delta OD/ng. Calculate intensity of the conjugated SUMO using the following formula: Sumoylation intensity protein ng mg protein = OD (sample negative control) slope x protein amount added (ug) 1000 Discover more at 14

16 DATA ANALYSIS Figure. 1. Illustrated standard curve generated using sumo protein control. Discover more at 15

17 RESOURCES 15. TROUBLESHOOTING Problem Cause Solution No signal for the sample Antibodies are not properly coated Ensure the concentration of the antibody solution Antibodies are not IP- Ensure the antibodies grade The protein sample is not properly extracted The protein amount is added into well insufficiently Nuclear extracts are stored incorrectly Reagents are added incorrectly Incubation time and temperature are incorrect Absence of sumoylation can be used for IP Ensure the protein extraction protocol is for nuclear protein extraction. Ensure extract contains a sufficient amount of protein Ensure the nuclear extracts are stored at -80 C Check if reagents are added in order and if some steps of the procedure were omitted by mistake Ensure the incubation time and temperature described in the protocol are followed correctly N/A Discover more at 16

18 RESOURCES High background present for the negative control background The negative control wells are contaminated with antibodies The wells are not sufficiently blocked with Blocking Buffer The well is not washed sufficiently Overdevelopment Ensure only negative control is added Increase blocking time to minutes Check if the wash at each step is performed according to the protocol Decrease development time Discover more at 17

19 RESOURCES 16. NOTES Discover more at 18

20 UK, EU and ROW Tel: +44-(0) Austria Tel: France Tel: Germany Tel: Spain Tel: Switzerland Tel (Deutsch): Tel (Français): US and Latin America Tel: ABCAM (22226) Canada Tel: China and Asia Pacific Tel: ( 中 國 聯 通 ) Japan Tel: +81-(0) Copyright 2014 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. RESOURCES All information / detail is correct at time of going to print. 19

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