Aldehyde Site Detection Kit

Transcription

1 Aldehyde Site Detection Kit Catalog Number KA assays Version: 04 Intended for research use only

2 Table of Contents Introduction... 3 Background... 3 Principle of the Assay... 3 General Information... 4 Materials Supplied... 4 Storage Instruction... 4 Materials Required but Not Supplied... 4 Precautions for Use... 5 Assay Protocol... 6 Reagent Preparation... 6 Sample Preparation... 6 Assay Procedure... 7 Data Analysis Performance Characteristics Resources Trouble shooting References Plate Layout KA / 13

3 Introduction Background Oxidative damage occurs in all living organisms from reactive oxygen species (ROS), which are a consequence of normal body processes such as metabolism. ROS react with proteins, resulting in protein modification, such as introduction of carbonyl groups into the protein. The modified proteins are dysfunctional and can be removed through degradation. Both mitochondrial DNA and nuclear DNA are constantly exposed to oxygen radicals, causing extensive oxidative damage. DNA damage by ROS has significant consequences since it causes mutations and genomic instability. Studies have shown that oxidative DNA damage accumulates with aging. Oxidative DNA damage has been implicated to be important in many diseases, including cancer. 1 Assessment of this damage in various biological matrices is essential for understanding the mechanisms of oxidative damage and its biological effects. Over the past decades, many analytical techniques, such as LC- and GC-mass spectrometry, have been developed to measure oxidative DNA base damage. More recently, antibody-based immunoassays, assays involving the use of DNA repair glycosylases, such as the comet assay, and slot blot assays using an aldehyde reactive probe (ARP), such as O-(biotinylcarbazoylmethyl) hydroxylamine, have been used to assess oxidative DNA damage. 2,3 The ARP probe reacts specifically with aldehyde groups that result from protein or DNA modification (see Figure 1). By using an excess amount of ARP, aldehyde sites in both protein and DNA can be converted to biotin-tagged aldehyde sites, which can be detected using avidin-conjugated reporters. Principle of the Assay The Aldehyde Site Detection Kit employs an ARP, O-(biotinylcarbazoylmethyl) hydroxylamine, as a probe to detect aldehyde sites in cells. Epigallocatechin Gallate (EGCG), a compound known to react with culture medium to generate hydrogen peroxide (H 2 O 2 ) and cause DNA damage, 4,5 is included to be used as a positive control. The kit is easy to use and can be easily adapted to high throughput screening for compounds imposing oxidative stress on cells or organisms. KA / 13

4 General Information Materials Supplied List of component Component Amount Aldehyde Site Assay Reactive Probe 120 μl Cell-Based Assay Buffer (10X) 50 ml Cell-Based Assay Fixative 12 ml Cell-Based BSA Blocking Solution 10 ml Aldehyde Site Assay Denaturing Solution 5 ml x 2 Cell-Based Assay Avidin-FITC Complex 1 vial Cell-Based Assay Epigallocatechin Gallate (EGCG) 1 vial Storage Instruction Component Storage Aldehyde Site Assay Reactive Probe -20 C Cell-Based Assay Buffer (10X) Room Temperature Cell-Based Assay Fixative Room Temperature Cell-Based BSA Blocking Solution 4 C Aldehyde Site Assay Denaturing Solution Room Temperature Cell-Based Assay Avidin-FITC Complex -20 C Cell-Based Assay Epigallocatechin Gallate (EGCG) -20 C Note: Avidin-FITC is light sensitive. Do not expose to direct intense light. Materials Required but Not Supplied Adjustable pipettes and a repeat pipettor. Triton X-100. A source of UltraPure water. Water used to prepare buffers must be deionized and free of trace organic contaminants ( UltraPure ). Use activated carbon filter cartridges or other organic scavengers. Glass distilled water (even if double distilled), HPLC-grade water, and sterile water (for injections) are not adequate. 6-, 12-, 24-, or 96-well plates for culturing cells. A plate centrifuge. Flow cytometer, fluorescence microscope, or plate reader equipped with laser/fluorescent filters capable of detecting FITC excitation and emission wavelengths of 485 and 535 nm, respectively. KA / 13

5 Precautions for Use WARNING: This product is for laboratory research use only: not for administration to humans. Not for human or veterinary diagnostic or therapeutic use. Please read the instructions carefully before beginning this assay. KA / 13

6 Assay Protocol Reagent Preparation NOTE: Water used to prepare all buffers must be deionized and free of trace organic contaminants ( UltraPure ). Use activated carbon filter cartridges or other organic scavengers. Glass distilled water (even if double distilled), HPLC-grade water, and sterile water (for injections) are not adequate. Assay Buffer Preparation To prepare an Assay Buffer solution, dilute 50 ml of Cell-Based Assay Buffer (10X) with 450 ml of UltraPure water. The diluted assay buffer will be stable for six months at room temperature. Wash Buffer Preparation To prepare a Wash Buffer solution, add 100 μl of Triton X-100 to 100 ml of diluted Assay Buffer, prepared above. The diluted wash buffer will be stable for six months at room temperature. Aldehyde Reactive Probe (ARP) Binding Solution Preparation Prior to use, thaw the Aldehyde Site Assay Reactive Probe on ice. To prepare a Binding Solution, dilute the reagent 1:200 in diluted Assay Buffer, prepared above and mix well. Prepare this solution just before use. Avidin-FITC Staining Solution Preparation Prior to use, reconstitute the whole vial of Cell-Based Assay Avidin-FITC Complex with 1 ml of diluted Assay Buffer, prepared above. The reconstituted Avidin-FITC Complex will be stable for one month at 4 C. To prepare a Staining Solution, dilute the reconstituted Avidin-FITC Complex prepared above 1:20 in diluted Assay Buffer, prepared above. Mix well and keep on ice. Protect from light. Prepare fresh Staining Solution for each assay. Sample Preparation Note: 1. Avidin-FITC is light sensitive. All staining procedures must be performed without direct exposure to intense light. Incubations should be done in the dark. 2. For all assay protocols described below, it is imperative that samples be analyzed immediately following completion of the staining. KA / 13

7 Treatment of the Cells 1. Culture cells in a 6-, 12-, 24-, or 96-well plates at a density of 5 x 10 5 cells/ml; grow cells overnight. 2. The next day, treat cells with experimental compounds or vehicle for 24 hours, or for the period of time required for your typical experimental protocol. To use the included EGCG as a positive control, dilute the EGCG 1:100-1:200 into your culture medium. 3. Terminate the experiment and examine aldehyde sites using the following staining procedure. Assay Procedure Flow Cytometry Fix Cells: 1. Collect the cells in a test tube and centrifuge at 400 x g for three minutes. Aspirate the supernatant. 2. Resuspend the cells at a density of 10 6 cells/ml in Cell-Based Assay Fixative. Mix well to ensure separation of individual cells. Incubate the cells in the Fixative for 10 minutes. 4. Wash the cells in 2-4 ml of Wash Buffer three times. Label ARP Sites: 1. Centrifuge the cells at 400 x g for three minutes and aspirate the supernatant. 2. Resuspend the cells at a density of cells/ml in the ARP Binding Solution and incubate for one hour at 37 C. Alternatively, incubate the cells overnight at 4 C and continue to the next step the following day. 4. Wash the cells in 2-4 ml of Wash Buffer three times. Denature DNA: 1. Centrifuge the cells at 400 x g for three minutes and aspirate the supernatant. 2. Resuspend the cells at a density of cells/ml in the Aldehyde Site Assay Denaturing Solution and incubate for 30 minutes at 37 C. 4. Wash the cells in 2-4 ml of Wash Buffer three times. Block: 1. Centrifuge the cells at 400 x g for three minutes and aspirate the supernatant. 2. Resuspend the cells at a density of cells/ml in the Cell-Based BSA Blocking Solution and incubate for 30 minutes at room temperature. KA / 13

8 Stain: 1. Centrifuge the cells at 400 x g for three minutes and aspirate the supernatant. 2. Resuspend the cells at a density of cells/ml in the Avidin-FITC Staining Solution prepared above and incubate for one hour at 37 C. 4. Wash the cells in 2-4 ml of Wash Buffer once. Analyze: 1. Resuspend the cells at a density of cells/ml in Wash Buffer. Mix well to ensure separation of individual cells. 2. The cells must be analyzed immediately. Oxidative damage recognized by ARP and labeled by Avidin-FITC can be detected in the FL1 channel. Fluorescence Microscopy Fix Cells: 1. Centrifuge the plate at 400 x g for three minutes. Aspirate the supernatant. 2. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of Cell-Based Assay Fixative to each well of a 6-, 12-, 24-, or 96-well plate, respectively. Incubate the cells in the Fixative for 10 minutes. 3. Centrifuge the plate at 400 x g for three minutes and aspirate the supernatant. 4. Wash the cells in Wash Buffer three times. Label ARP Sites: 2. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of the ARP Binding Solution to each well of a 6-, 12-, 24-, or 96-well plate, respectively and incubate for one hour at 37 C. Alternatively, incubate the cells overnight at 4 C and continue to the next step the following day. 4. Wash the cells in Wash Buffer three times. Denature DNA: 2. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of the Aldehyde Site Assay Denaturing Solution to each well of a 6-, 12-, 24-, or 96-well plate, respectively and incubate for 30 minutes at 37 C. 3. Centrifuge the plate at 400 x g for three minutes and aspirate the supernatant. 4. Wash the cells in Wash Buffer three times. Block: 2. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of the Cell-Based BSA Blocking Solution to each well of a 6-, 12-, 24-, KA / 13

9 or 96-well plate, respectively and incubate for 30 minutes at room temperature. Stain: 2. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of the Avidin-FITC Staining Solution prepared above to each well of a 6-, 12-, 24-, or 96-well plate, respectively and incubate for one hour at 37 C. 4. Wash the cells in Wash Buffer once. Analyze: 1. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of Wash Buffer to each well of a 6-, 12-, 24-, or 96-well plate, respectively. 2. The cells must be analyzed immediately. Oxidative damage recognized by ARP and labeled by Avidin-FITC can be detected with a fluorescent filter designed to detect fluorescein (excitation/emission = 485/535). Plate Reader Fluorescence Detection A 96-well BLACK culture plate should be used for this method. Fix Cells: 1. Centrifuge the plate at 400 x g for three minutes. Aspirate the supernatant. 2. Add 50 μl of Cell-Based Assay Fixative to each well of the plate and incubate the cells in the Fixative for 10 minutes. 3. Centrifuge the plate at 400 x g for three minutes and aspirate the supernatant. 4. Wash the cells in Wash Buffer three times. Label ARP Sites: 2. Add 50 μl of the ARP Binding Solution to each well of the plate and incubate for one hour at 37 C. Alternatively, incubate the cells overnight at 4 C and continue to the next step the following day. 4. Wash the cells in Wash Buffer three times. Denature DNA: 2. Add 50 μl of the Aldehyde Site Assay Denaturing Solution to each well of the plate and incubate for 30 minutes at 37 C. 3. Centrifuge the plate at 400 x g for three minutes and aspirate the supernatant. 4. Wash the cells in Wash Buffer three times. KA / 13

10 Block: 2. Add 50 μl of the Cell-Based BSA Blocking Solution to each well of the plate and incubate for 30 minutes at room temperature. Stain: 2. Add 50 μl of the Avidin-FITC Staining Solution prepared above to each well of the plate and incubate for one hour at 37 C. 4. Wash the cells in Wash Buffer once. Analyze: 1. Add 50 μl of Wash Buffer to each well of the plate. 2. The cells can be analyzed with a fluorescence plate reader and must be analyzed immediately. Oxidative damage recognized by ARP and labeled by Avidin-FITC can be measured as fluorescence intensity with excitation and emission wavelength of 485 nm and 535 nm, respectively. KA / 13

11 Data Analysis Performance Characteristics Cell Staining Figure 1. Hydrogen peroxide induces oxidative damage in HeLa cells. HeLa cells were plated at a density of 1 x 10 4 cells/well in a 96-well plate. The next day, cells were treated with vehicle (control), 0.015% (4.9 mm) H 2 O 2, and 0.03% (9.8 mm) H 2 O 2 for four hours. Cells were then processed for staining according to the protocol described in the kit booklet. Panel A: cells treated with vehicle (control) had background levels of oxidative protein/dna damage, appearing as faint staining both in the cytoplasm and nuclei. Panel B: cells treated with 0.015% (4.9 mm) H 2 O 2 had elevated levels of oxidative damage in both the cytoplasm and nucleus, as evidenced by an increase in staining intensity. Panel C: cells treated with 0.03% (9.8 mm) H 2 O 2 had strong staining, indicating severe oxidative damage. Figure 2. EGCG induces oxidative damage in HeLa cells. HeLa cells were plated at a density of 1 x 10 4 cells/well in a 96-well plate. The next day, cells were treated with vehicle (control), 100 μm EGCG, and 200 μm EGCG and incubated overnight. Previous experiments have demonstrated that EGCG reacts with culture medium to generate significant amounts of H 2 O 2. 4 Cells were then processed for staining according to the protocol described in the kit booklet. Panel A: cells treated with vehicle (control) had background levels of oxidative damage, shown as faint staining both in the cytoplasm and nuclei. Panel B: cells treated with 100 μm EGCG had elevated levels of oxidative DNA, as evidenced by an increase in staining intensity. Panel C: cells treated with 200 μm EGCG had strong staining in both the cytoplasm and nuclei, indicating severe oxidative damage. KA / 13

12 Resources Trouble shooting Problem Possible Causes Recommended Solutions No staining, including positive control. Control cells without treatment show strong staining. A. Samples were not well permeabilized. B. Reagents are not added in right order Control cells are not healthy. A. After fixation, incubate cells in the Assay Buffer for five minutes before processed for next step. B. Add reagents in right order. Use only healthy cells. References 1. Cooke, M.S., Evans, M.D., Dizdaroglu, M., et al. Oxidative DNA damage: Mechanisms, mutation, and disease. FASEB J. 17, (2003). 2. Nakamura, J., Walker, V.E., Upton, P.B., et al. Highly sensitive apurinic/apyrimidinic site assay can detect spontaneous and chemically induced depurination under physiological conditions. Cancer Res. 58, (1998). 3. Poulsen, H.E., Weimann, A., and Loft, S. Methods to detect DNA damage by free radicals: Relation to excercise. Proceedings of the Nutrition Society 58, (1999). 4. Elbling, L., Weiss, R.-M., Teufelhofer, O., et al. Green tea extract and (-)-epigallocatechin-3-gallate, the major tea catechin, exert oxidant but lack antioxidant activities. FASEB J. 19(7), (2005). 5. Tachibana, H., Molecular basis for cancer chemoprevention by green tea polyphenol EGCG. Forum Nutr. 61, (2009). KA / 13

13 A B C D E F G H Plate Layout KA / 13