OxiSelect™ UV-Induced DNA Damage ELISA Kit
Contents
1. Product Manual OxiSelect UV Induced DNA Damage ELISA Kit CPD Quantitation Trial Size Catalog Number STA 322 T 32 assays FOR RESEARCH USE ONLY Not for use in diagnostic procedures CELL BIOLABS INC leoman Qal Atan Trag om Sosa Daca Creating Solutions for Life Science Research Introduction Absorption of ultraviolet UV light produces two predominant types of DNA damage cyclobutane pyrimidine dimers CPD and pyrimidine 6 4 pyrimidone photoproducts 6 4PP Figure 1 The result is a transition of C to T and CC to TT which are the most frequent mutations of p53 in both human and mouse skin cancers UV damaged DNA is usually repaired by nucleotide excision repair NER or base excision repair BER After UV exposure cells activate p53 and stall the cell cycle for repair If the damage is too severe the cell will trigger apoptosis to get rid of DNA damaged potentially mutant cells Cell Biolabs OxiSelect Oxidative UV induced DNA Damage ELISA Kit CPD Quantitation 1s an enzyme immunoassay developed for rapid detection and quantitation of CPD in any DNA samples The quantity of CPD in unknown sample is determined by comparing its absorbance with that of a known CPD DNA standard curve This Trial Size kit provides sufficient reagents to perform up to 32 assays including standard curve and unknown samples O N N dipyrimidines g lt 6 4 photoproduct Figure 1 Structures of DNA lesions induced by UV Li2. 25 pg mL to single stranded DNA by incubating the DNA sample at 95 C for 10 minutes and rapidly chilling on ice for 10 minutes Note Aliquot and store denatured CPD DNA standard at 20 C Repeat the above denaturation step every time you prepare the CPD DNA standard 2 Dilute desired amount of freshly denatured DNA sample 10 fold to 2 5 ug mL in cold PBS For example add 10 uL of the 25 ug mL CPD DNA standard to 90 uL of cold PBS Prepare a dilution series of CPD DNA standards in the concentration range of 0 ng mL 250 ng mL by diluting the denatured CPD DNA Standard in cold PBS according to Table 1 below 2 5 pg mL Denatured CPD DNA Standard Tubes CPD DNA Standard uL Cold PBS uL ng mL 400 of Tube 1 400 of Tube 2 oo 0 Table 1 Preparation of CPD DNA Standards CELL BIOLABS INC Tha i Assay Protocol l Extract DNA from cell or tissue samples using a commercial DNA Extraction kit or other desired method Convert DNA sample to single stranded DNA by incubating the sample at 95 C for 10 minutes and rapidly chilling on ice for 10 minutes Dilute denatured DNA sample to 2 ug mL or less in cold PBS 4 Add 100 uL of unknown denatured DNA sample or CPD DNA standards to the wells of the DNA 10 11 12 13 High Binding plate Incubate at 37 C for 2 hours or overnight at 4 C Each DNA sample including unknown and standard should be assayed in duplicate Remove the DNA solutions and wash t
3. M et al 2015 Standardized 3D bioprinting of soft tissue models with human primary cells J Lab Autom doi 10 1177 2211068214567146 Donninger H et al 2015 The RASSF1A tumor suppressor regulates XPA mediated DNA repair Mol Cell Biol 35 277 287 Zirkin S et al 2013 The PIM 2 Kinase is an essential component of the ultraviolet damage response that acts upstream to E2F 1 and ATM J Biol Chem 288 21770 21789 Burgess H M et al 2011 Nuclear relocalisation of cytoplasmic poly A binding proteins PABPI and PABP4 in response to UV irradiation reveals mRNA dependent export of metazoan PABPs J Cell Sci 124 3344 3355 Warranty These products are warranted to perform 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 Contact Information Cell Biolabs Inc 7758 Arjons Drive San Diego CA 92126 Worldwide 1 858 271 6500 USA Toll Free 1 888 CBL 0505 E mail tech cellbiolabs com www cellbio
4. One 100 uL tube Assay Diluent Part No 310804 T One 20 mL bottle 10X Wash Buffer Part No 310806 T One 30 mL bottle Substrate Solution Part No 310807 T One 4 mL amber bottle Stop Solution Part No 310808 T One 4 mL bottle CPD DNA Standard Part No 232203 T One 40 uL vial of 25 ug mL CPD DNA in 1X TE Buffer Materials Not Supplied oo OA e ee a DNA samples such as cell or tissue genomic DNA DNA Extraction Kit Heating Block PBS 10 uL to 1000 uL adjustable single channel micropipettes with disposable tips 50 uL to 300 uL adjustable multichannel micropipette with disposable tips Multichannel micropipette reservoir Microplate reader capable of reading at 450 nm 620 nm as optional reference wave length CELL BIOLABS INC Tha i Storage Upon receipt store the CPD DNA standard at 20 C and all other components at 4 C until their expiration dates Preparation of Reagents e 1X Wash Buffer Dilute the 10X Wash Buffer to 1X with deionized water Stir to homogeneity e 1X Blocking Reagent Prepare the appropriate volume for the number of samples being tested Immediately prior to using dilute the provided 100X Blocking Reagent 1 100 in 1X PBS Do not store e Anti CPD Antibody and Secondary Antibody Immediately before use dilute the Anti CPD Antibody 1 1000 and Secondary Antibody 1 1000 with Assay Diluent Do not store diluted solutions Preparation of Standard Curve 1 Convert CPD DNA standard
5. eader using 450 nm as the primary wave length Example of Results The following figures demonstrate typical Oxidative UV induced DNA Damage ELISA CPD Quantitation results One should use the data below for reference only This data should not be used to interpret actual results h J erage et E in TI m O OD 450nm 10 20 30 UV Light Treatment min Figure 3 DNA Damage Induced by UV Light 0 2 mg mL Calf thymus DNA was exposed to UV light inside a cell culture hood for the time indicated The CPD levels in 40 ng denatured DNA samples were determined as described in the Assay Protocol N CELL BIOLABS INC Creating Solutions for Life Science Research References l Lippke JA Gordon LK Brash DE Haseltine WA 1981 Proc Natl Acad Sci U S A 78 3388 3392 Mitchell DL Nairn RS 1989 Photochem Photobiol 49 805 819 Ananthaswamy HN Loughlin SM Cox P Evans RL Ullrich SE Kripke ML 1997 Nat Med 3 510 514 Soehnge H Ouhtit A Ananthaswamy ON 1997 Front Biosci 2 D538 D551 el Deiry WS Tokino T Velculescu VE Levy DB Parsons R Trent JM Lin D Mercer WE Kinzler KW Vogelstein B 1993 Cell 75 817 825 Hermeking H Lengauer C Polyak K He TC Zhang L Thiagalingam S Kinzler KW Vogelstein B 1997 Mol Cell 1 3 11 Hill LL Ouhtit A Loughlin SM Kripke ML Ananthaswamy HN Owen Schaub LB 1999 Science 285 898 900 Recent Product Citations l 2 Rimann
6. ght Assay Principle CDP DNA standards or unknown DNA samples are first heat denatured before adsorbed onto a DNA high binding plate The CPDs present in the sample or standard are probed with an anti CPD antibody followed by an HRP conjugated secondary antibody The CPD content in an unknown sample is determined by comparing with a standard curve that is prepared from predetermined CPD DNA standards 2 i CELL BIOLABS INC Related Products es eS se a STA 320 OxiSelect Oxidative DNA Damage ELISA Kit 8 OHdG Quantitation STA 321 OxiSelect DNA Double Strand Break DSB Staining Kit STA 323 OxiSelect UV induced DNA Damage ELISA Kit 6 4PP Quantitation STA 324 OxiSelect Oxidative DNA Damage Quantitation Kit AP sites STA 325 OxiSelect Oxidative RNA Damage ELISA Kit 8 OHG Quantitation STA 326 OxiSelect Cellular UV induced DNA Damage ELISA Kit CPD STA 327 OxiSelect Cellular UV induced DNA Damage Staining Kit CPD STA 328 OxiSelect Cellular UV induced DNA Damage ELISA Kit 6 4PP STA 329 OxiSelect Cellular UV induced DNA Damage Staining Kit 6 4PP Kit Components l e st a a ee DNA High Binding Plate Part No 232201 T One strip well plate containing 32 wells 8 x 4 precoated with DNA binding matrix Anti CPD Antibody Part No 232202 T One 10 uL vial of anti CPD Secondary Antibody HRP Conjugate Part No 10902 One 50 uL vial Blocking Reagent 100X Part No 232206 T
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8. wice with PBS Blot plate on paper towels to remove excess fluid Add 150 uL of Assay Diluent to each well and block for 1 hour at room temperature Aspirate the Assay Diluent from the wells and add 100 uL of the diluted anti CPD antibody to each well and incubate at room temperature for hour on an orbital shaker Wash microwell strips 3 times with 250 uL 1X Wash Buffer per well with thorough aspiration between each wash After the last wash empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1X Wash Buffer Add 150 uL of prediluted 1X Blocking Reagent to each well see Preparation of Reagents Section Incubate the plate for 60 minutes at room temperature on an orbital shaker Wash microwell strips 3 times according to step 7 above Add 100 uL of the diluted Secondary Antibody Enzyme Conjugate to each well and incubate at room temperature for hour on an orbital shaker Wash microwell strips 3 times according to step 7 above Proceed immediately to the next step Warm Substrate Solution to room temperature Add 100 uL of Substrate Solution to each well including the blank wells Incubate at room temperature on an orbital shaker Actual incubation time may vary from 2 30 minutes Stop the enzyme reaction by adding 100 uL of Stop Solution into each well including the blank wells Results should be read immediately color will fade over time Read absorbance of each microwell on a standard microplate r
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