DNA damage - AP site- Assay Kit (Cell-Based)

Transcription

1 ab DNA damage - AP site- Assay Kit (Cell-Based) Instructions for Use For the detection of aldehyde sites in cells. This product is for research use only and is not intended for diagnostic use.

2 1

3 Table of Contents 1. Overview 3 2. Background 4 3. Components and Storage 5 4. Pre-Assay Preparation 7 5. Assay Protocol 8 6. Data Analysis Troubleshooting 17 2

4 1. Overview ab 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, 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. Figure 1. Mechanism of ARP binding at an aldehyde group 3

5 2. 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. 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. 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. 4

6 3. Components and Storage For best results, remove components and store as stated below. Item Quantity Storage Aldehyde Site Assay Reactive Probe 1 vial -20 C Aldehyde Site Assay Buffer (10X) 1 vial RT Cell-Based Assay Fixative 1 vial RT Aldehyde Site Assay Blocking Solution 1 vial 4 C Aldehyde Site Assay Denaturing Solution 2 vials RT Aldehyde Site Assay Avidin-FITC Complex 1 vial -20 C Cell-Based Assay Epigallocatechin Gallate (EGCG) 1 vial -20 C 5

7 Materials Needed 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. 6

8 4. Pre-Assay 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. A. Assay buffer preparation To prepare an Assay Buffer solution, dilute 50 ml of Aldehyde Site Assay Buffer (10X) with 450 ml of UltraPure water. The diluted Assay Buffer will be stable for six months at room temperature. B. 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. C. 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. 7

9 D. Avidin-FITC Staining Solution Preparation Prior to use, reconstitute the whole vial of Aldehyde Site 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. 5. Assay Protocol A. 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 Cell-Based Assay Epigallocatechin Gallate(EGCG) 1:100-1:200 into your culture medium. 3. Terminate the experiment and examine aldehyde sites using the following staining procedure. 8

10 B. Flow cytometry Fix Cells: 1. Collect the cells in a test tube and centrifuge at 400 x g for three minutes. Aspirate 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. 3. Centrifuge the cells at 400 x g for three minutes and aspirate 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 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. 3. Centrifuge the cells at 400 x g for three minutes and aspirate 9

11 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 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. 3. Centrifuge the cells at 400 x g for three minutes and aspirate 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 2. Resuspend the cells at a density of cells/ml in the Aldehyde Site Assay Blocking Solution and incubate for 30 minutes at room temperature. Stain: 1. Centrifuge the cells at 400 x g for three minutes and aspirate 10

12 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. 3. Centrifuge the cells at 400 x g for three minutes and aspirate 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. 11

13 C. Fluorescence Microscopy 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 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 4. Wash the cells in Wash Buffer three times. Label ARP Sites: 1. Centrifuge the plate at 400 x g for three minutes and aspirate 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. 12

14 3. Centrifuge the cells at 400 x g for three minutes and aspirate 4. Wash the cells in Wash Buffer three times. Denature DNA: 1. Centrifuge the plate at 400 x g for three minutes and aspirate 2. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of the 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 4. Wash the cells in Wash Buffer three times. Block: 1. Centrifuge the plate at 400 x g for three minutes and aspirate 2. Add 1 ml, 0.5 ml, 250 μl, or 50 μl of the Aldehyde Site Assay Blocking Solution to each well of a 6-,12-,24-, or 96-well plate, respectively and incubate for 30 minutes at room temperature. 13

15 Stain: 1. Centrifuge the plate at 400 x g for three minutes and aspirate 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. 3. Centrifuge the cells at 400 x g for three minutes and aspirate 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). 14

16 6. Data Analysis A. Performance Characteristics Figure 2. 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 this 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. 15

17 Panel C: cells treated with 0.03% (9.8 mm) H 2 O 2 had strong staining, indicating severe oxidative damage. Figure 3. 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. Cells were then processed for staining according to the protocol described in this 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 the cytoplasm and nuclei, indicating severe oxidative damage. 16

18 7. Troubleshooting 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 A. 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 A. Use only healthy cells 17

19 18

20 UK, EU and ROW Tel: +44 (0) US, Canada and Latin America Tel: ABCAM (22226) China and Asia Pacific Tel: ( 中國聯通 ) Japan technical@abcam.co.jp Tel: +81-(0) Copyright 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark. All information / detail is correct at time of going to print. 19