TACS Apoptotic DNA Laddering Kit

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1 Instructions for Use For Research Use ONLY TACS Apoptotic DNA Laddering Kit This Protocol Covers the Following Kits: Cat# ET Ethidium bromide 20 reactions Cat# K Radioactive 20 reactions Cat# K Chemiluminescent 20 reactions Cat# K Colorimetric 20 reactions Cat# K Tissue Supplement 20 reactions Trevigen's TACS Apoptotic DNA laddering kits are used to assay cells and tissues for apoptosis by detecting double-strand DNA breaks, and determining the level of DNA degradation 1. DNA fragmentation is considered to be the hallmark of apoptosis. During apoptosis, DNA is cleaved into internucleosomally sized (~ base pair) fragments. These DNA fragments may be extracted from cells and analyzed by horizontal gel electrophoresis followed by ethidium bromide staining. Often, however, the number of cells available for analysis, or the amount of apoptotic cells within a sample, are insufficient to be visualized using ethidium bromide. To address this, Trevigen developed the Radioactive, Chemiluminescent, and Colorimetric kits to enhance the sensitivity of the apoptotic laddering assay. When analyzing tissue samples, it is necessary to disrupt the tissue and create a cell suspension. The Tissue Supplement provides the reagents to allow DNA laddering analysis in tissues. REFERENCES: 1. Rosl, F., 1992, "A simple and rapid method for detection of apoptosis in human cells," Nucleic Acids Research 20(19): FEATURES: Complete kits: extraction, labeling, detection reagents, and gel matrix included. Detect as little as 2% apoptotic DNA laddering in 10 5 cells. Rapid DNA isolation. Isotopic or non-isotopic labeling options. 20 sample size per kit. (Non-isotopic kits provide sufficient reagents for 4 nylon membranes with 5 samples each). Trevigen, Inc Helgerman Court, Gaithersburg, MD USA Voice: TREVIGEN ( ) FAX: info@trevigen.com TREVIGEN is a registered trademark and TACS, TreviGel, PeroxyGlow are trademarks of Trevigen, Inc Trevigen, Inc., All Rights Reserved -1-

2 KIT COMPONENTS: Cat# Component Volume Concentration Storage Conditions ET (ETBR) K (RAD.) K (CHEM.) K (COLOR.) K (TISS.) Lysis Solution 1 2 ml N/A Room temperature Extraction Solution 2 20 ml N/A Room temperature Extraction Buffer 3 8 ml N/A Room temperature Sodium Acetate 4 1 ml N/A Room temperature DNase-free Water 5 2 ml N/A Room temperature Sample Buffer 2 ml N/A Room temperature DL 10X Klenow Buffer 0.5 ml 10X -20 C DL Klenow Enzyme 20 µl 5 U/µl -20 C DL Klenow Water 1 ml N/A -20 C Gel Loading Buffer 250 µl N/A Room temperature P TreviGel 500 Powder 6 g N/A Room temperature DL dntp Mix 40 µl N/A -20 C DL Strep-HRP 40 µl 100 µg/µl 4 C (Do not freeze) DL Control DNA 4 µl 0.5 µg/µl -20 C Chemiluminescent ( K) PeroxyGlow A 20 ml N/A 4 C PeroxyGlow B 20 ml N/A 4 C Colorimetric ( K) Blue Membrane Soln 40 ml N/A 4 C Supplement Tissue Extraction Reagents ( K) X Tissue Buffer 0.5 ml N/A -20 C TACS DNA Laddering biotinylated deoxynucleotide mix MATERIALS NOT SUPPLIED: α- 32 P-dATP or α- 32 P-dCTP (3000 Ci mmol -1 ) 2-propanol (isopropanol) 70% ethanol microcentrifuge tubes, 1.5 ml pipet tips (1-200 µl) gloves (i.e. latex) 1, 5 and 10 ml serological pipets plastic wrap for exposure of gel during autoradiography autoradiographic film and supplies 0.5 M ethylenediaminotetraacetic acid (EDTA), ph 8 (OPTIONAL) Reagents for removal of unincorporated nucleotides (OPTIONAL) 10X PBS (Cat# ) 50X TAE (Cat# ) Powdered Milk or Bovine Serum Albumin uncharged Nylon Membrane 20X SSC Concentrated hydrochloric acid 5 M Sodium Hydroxide 5 M Sodium Chloride 1 M Tris, ph 7.5 Deionized water EQUIPMENT REQUIRED: microcentrifuge electrophoresis setup and power supply 65 C water bath Adjustable pipetors: 1-20 µl µl µl -20 C freezer for storage of components vortex mixer UV spectrophotometer (OPTIONAL) -2-

3 I. General Notes: It is recommended that you proceed with the entire protocol without stopping. If necessary, samples may be frozen at -20 or -80 C after steps 3 or 9 of the DNA Isolation procedure. Cell suspensions prepared from tissue using the Tissue Extraction Kit ( K) may be stored by freezing after step 3 of that procedure. If processing cells from culture, proceed to DNA Isolation from Cultured Cells below. II. DNA Isolation from Tissues: (For best results, process the tissue sample as soon as possible after removal from the animal.) N.B. - While additional Tissue Processing Reagents are supplied for this portion of the kit, the original DNA Laddering Kit is limited to processing 20 samples of 100 µl. 1. Mince the tissue into smaller pieces and freeze in liquid nitrogen. 2. Grind the frozen tissue samples into powder using either a hammer or mortar and pestle (these should be pre-chilled to avoid thawing of the sample). Add additional liquid nitrogen if required. Process 0.2 to 1 gram of the powder at one time. Please consider the amount of material being processed, as only 20 x 100 µl samples can be processed in the DNA Laddering Kit. 3. Suspend the tissue into Sample Buffer in the following ratio: 0.1 g of powdered tissue sample 200 µl of Sample Buffer (Cat# ) 20 µl of 10X Tissue Buffer (Cat# ) Allow the tissue to thaw and incubate at 50 C for 12 to 18 hours. Gentle shaking is recommended. 4. Transfer 100 µl of the cell suspension from step 3 to a new microcentrifuge tube. Add 100 µl Lysis Solution 1 (Cat# ) provided with the DNA Laddering Kit and mix thoroughly by inverting tube several times. NOTE: The remaining cell suspension may be frozen at -80 C for future use. Continue with step 4 (indicated by a ) of the DNA ISOLATION FROM CULTURED CELLS Protocol below. III. DNA Isolation from Cultured Cells: (For best results, process samples immediately after harvesting.) 1. Suspension Cells: Harvest cells (10 5 to 10 7 ) by centrifugation and resuspend pellet in 100 µl Sample Buffer (10 6 to 10 8 cells ml -1 ). Proceed to step 2. Adherent Cells: Lyse cells directly in culture dish by adding 100 µl of Sample Buffer and 100 µl Lysis Solution 1. Gently scrape the cells or pipet until completely in solution. Proceed to step 4 below. 2. Incubate this cell suspension at room temperature for 5 to 15 minutes. 3. Lyse the sample and stabilize the DNA by adding 100 µl of Lysis Solution 1. Mix thoroughly by inverting tube several times. 4. Make sure that the sample is now in a ml microcentrifuge tube. Shake Extraction Solution 2, then add 700 µl to the sample. 5. Add 400 µl of Extraction buffer 3 to the sample. Vortex for 10 seconds. -3-

4 6. Microcentrifuge at 12,000 xg for 5 minutes. 7. Transfer the upper layer (aqueous) to a new microcentrifuge tube. Avoid removing the darker, lower organic or interface layer. If the recovered aqueous layer is cloudy repeat steps 6 and 7. Discard the organic layer by protocols established in your laboratory. NOTE: If the aqueous layer is very cloudy, a chloroform extraction may be required at this point. This can be performed by adding 400 µl of chloroform to the aqueous layer (Use of a fume hood or good ventilation is suggested). Vortex the sample for 10 seconds, and repeat steps 6 and 7. When removing the upper phase: Upper Aqueous Phase Interphase (avoid) Lower Organic Phase Carefully remove the upper aqueous phase without disturbing the interphase. If you accidentally remove the interphase, centrifuge the tube again, and carefully remove the upper layer. 8 Add 40 µl (i.e. 0.1 volume) of Sodium Acetate 4 to the aqueous sample that was transferred. Mix by inverting. 9. Add 440 µl (i.e. equal volume) of 2-propanol and mix by inverting. N.B. - Optional stopping point by placing samples at -20 C overnight. 10. Microcentrifuge at 12,000 xg for 10 minutes at room temperature. 11. Carefully remove the supernatant without disturbing the pellet. Add 1 ml of 70% ethanol to the pellet. Mix gently by inverting the tube several times. 12. Microcentrifuge at 12,000 xg for 5 minutes at room temperature. 13. Carefully remove the supernatant without disturbing the pellet. Allow the pellet to dry. The pellet can be dried by simply inverting the tube on a laboratory tissue and allowing the liquid to evaporate. The pellet can also be dried with the aid of a vacuum centrifuge apparatus. 14. Resuspend the pellet in 100 µl of DNase-free Water Quantitate the DNA spectrophotometrically. Take 5 µl of the DNA sample and add to 1 ml of water. Determine the optical density at 260 nm against a water blank. The DNA concentration of the undiluted sample can be determined as: concentration (µg/µl) = Absorbance 260 of diluted sample x 9.88* * The 9.88 is a multiplication factor that accounts for the 1:200 dilution and the O.D. of the DNA in H 2 0. Alternatively, the DNA concentration can be estimated from an ethidium bromide stained gel by comparing with a sample of DNA of known concentration. 16. Proceed based upon the kit you are using: ET, Ethidium Bromide Kit: Ethidium Bromide procedure, page K, Isotopic Kit: Radiolabeling of DNA procedure, page K, Chemiluminescent Kit: Labeling of DNA procedure, Bottom of page K, Colorimetric Kit: Labeling of DNA procedure, Bottom of page

5 IV. Ethidium Bromide Kit: 1. To 1 to 2 µg of isolated DNA add sufficient water (DNase free) to make volume up to 10 µl. Add 2 µl of 5X Gel Loading Buffer (provided with kit). Load onto a 1.5% TreviGel 500 gel in 1X TAE buffer. For gel preparation, refer to the end of this protocol "Casting TreviGel 500", on page 7. Separate the DNA fragments by electrophoresis (typically at 100 volts) until the bromophenol blue dye front reaches the lower third of the gel. 2. Remove the gel from the apparatus and immerse gel in 0.5 µg/ml ethidium bromide prepared in H 2 O or 1X TAE buffer for 15 minutes. CAUTION: Wear gloves when handling solutions or gels containing ethidium bromide. Visualize DNA by illumination on a UV light source. CAUTION: Wear protective goggles and clothing during exposure to UV Light. V. Isotopic Kit: 1. Set up labeling reaction with 0.5 to 1 µg of cellular DNA, as follows: 1X Cellular DNA X µl X is determined from the spectrophotometric calculations DL 10X Klenow buffer 1 µl DL Klenow water Y µl Y = 8 - X - Z radiolabeled nucleotide* Z µl Z is the volume required to make 0.5 µci of radiolabel DL Klenow enzyme 1 µl (dilution may be required) 10 µl * α- 32 P-dATP or α- 32 P-dCTP, 3000 Ci/mmol may be used. 2. Incubate at room temperature for 10 minutes. 3. Stop the reaction by adding 2 µl of the Gel Loading Buffer. Load sample** onto a 1.5% TreviGel 500 gel in 1X TAE buffer. For gel preparation, refer to the end of this protocol "Casting TreviGel 500", on page 7. Separate the DNA fragments by electrophoresis (typically at 100 volts) until the bromophenol blue dye front reaches the lower third of the gel. **NOTE: You may wish to load 7, 4, and 1 µl aliquots into sequential lanes rather than loading all of the sample into one lane. 4. After electrophoresis, cut-off the lower portion of the gel below the bromophenol blue dye front containing the unincorporated radiolabeled nucleotide, and discard appropriately. 5. Fix the gel by immersing into 200 ml of 10% acetic acid in water, and gently rocking for 1 hour. Repeat for one hour (to overnight) in 200 ml fresh 10% acetic acid. Dry gel onto Whatman 3MM paper using a gel dryer (set below 70 C), or a hair dryer, prior to autoradiography. Depending upon labeling efficiency, exposure times will vary from 1 hour to overnight. For rapid viewing, the undried gel can be wrapped in plastic and exposed to x-ray film directly (bands from dried gels are sharper). VI. Chemiluminescent and Colorimetric Kits A. Labeling of DNA: 1. Set up labeling reaction with 0.5 to 1 µg of cellular DNA (or 1 µl of DL Control DNA), as follows: 1X Cellular DNA X µl X is determined from the spectrophotometric calculations DL 10X Klenow buffer 1 µl DL Klenow water Y µl Y = 6 - X DL dntp mix 2 µl DL Klenow enzyme 1 µl 10 µl 2. Incubate at room temperature for 10 minutes. -5-

6 3. Stop the reaction by adding 2 µl of the Gel Loading Buffer. Load sample** onto a 1.5% TreviGel 500 gel in 1X TAE buffer. For gel preparation, refer to the end of this protocol "Casting TreviGel 500", on page 7. Separate the DNA fragments by electrophoresis (typically at 100 volts) until the bromophenol blue dye front reaches the lower third of the gel. **NOTE: You may wish to load 7, 4, and 1 µl aliquots into sequential lanes rather than loading all of the sample into one lane. B. Southern Transfer of DNA: The following protocol may be substituted with an appropriate DNA electroblotting procedure using UN- CHARGED nylon or nitrocellulose membrane. For orientation it is helpful to cut one corner of the gel before proceeding. 1. Depurinate by soaking in 500 ml of 0.25 M HCl for 15 minutes at room temperature. 2. Rinse twice with deionized water. 3. Soak gel in 500 ml of a solution containing 0.4 M NaOH and 0.8 M NaCl for 30 minutes at room temperature. 4. Soak gel in 500 ml of a solution containing 0.5 M Tris, ph 7.5 and 1.5 M NaCl for 30 minutes at room temperature. 5. Transfer DNA to an uncharged Nylon or nitrocellulose membrane by Southern transfer overnight using 10X SSC buffer (See diagram below). Southern Transfer Setup Absorbant Material (i.e. paper toweling or blotting paper) Whatman 3 MM Paper Wick Support Whatman 3 MM Paper Uncharged Nylon Membrane Gel Whatman 3 MM Paper 10X SSC Level Lab Dish (i.e. Pyrex glass baking dish) 6. Dismantle apparatus. Using a pencil mark the orientation of the gel and position of the wells. OPTIONAL: UV crosslink for 15 minutes. 7. Place membrane into 50 to 100 ml of 1X PBS containing 5% powdered milk (or 1-2% BSA) and shake/rock for 30 minutes at room temperature. Proceed with detection procedures below: OPTIONAL: It is helpful to check the efficiency of transfer by ethidium bromide staining the gel (0.5 µg/ml) and checking if any DNA was not transferred. Typically, large molecular weight DNA transfer is less efficient, but is sufficient for DNA laddering detection. C. Detection: a) Chemiluminescent Kit ( K) 1. Place the membrane in a plastic bag or dish with 20 ml 1X PBS. Add 10 µl of Strep-HRP conjugate. Shake/ rock at room temperature for 30 minutes. -6-

7 2. Wash the membrane three times, 5 minutes each wash, in 100 ml 1X PBS at room temperature. 3. In a clean tube, combine 5 ml of PeroxyGlow A and 5 ml PeroxyGlow B and mix well by inverting. This solution is stable for about 1 hour. Place the membrane into a container that is just larger than the membrane and add the PeroxyGlow solution. Shake/Rock the membrane for 1 to 5 minutes making sure to get an even distribution of the PeroxyGlow solution over the entire membrane. 4. Quick dry the membrane with paper toweling, but avoid completely drying the membrane. Immediately cover in plastic wrap. Expose the membrane to film for 15 seconds to 5 minutes and develop the autoradiograph. Adjust exposure times as necessary. b) Colorimetric Kit ( K) 1. Place the membrane in a plastic bag or dish with 20 ml 1X PBS. Add 10 µl of Strep-HRP conjugate. Shake/rock at room temperature for 30 minutes. 2. Wash the membrane three times, 5 minutes each wash, in 100 ml 1X PBS at room temperature. 3. Place membrane into a container just larger than the membrane and add 10 ml Blue Membrane Solution (BMS). Shake/rock the membrane until blue color is clearly developed. The color development should be complete within 10 minutes. Be sure to get an even distribution of the BMS over the membrane. 4. The membrane should be photographed with a yellow filter for a permanent record. The membrane should be stored in the dark to reduce fading. VII. Interpretation: A typical ladder pattern of multiples of approximately 200 base pairs is a hallmark of apoptosis in the majority of cell types. Although the method is not quantitative, qualitative comparisons can be made with control cells and between samples prepared from equivalent cell numbers. Faint DNA laddering patterns may be evident in control samples due to the low level of apoptosis that normally takes place in many cells in culture. The appearance of 2 distinct bands when using the Ethidium Bromide kit may indicate the presence of RNA contamination. Refer to the trouble shooting guide for information on removing RNA. VIII. Casting TreviGel 500 : TreviGel 500 powder can be used to prepare horizontal gels for standard laboratory purposes. The following method for TAE gel casting gives consistent, high resolution results. For high efficiency transfer out of gels, thinner gels are recommended (Transfer efficiency α 1/distance), therefore, it is preferable to use one-half the volume typically used in your gel apparatus. 1. Weigh out enough TreviGel 500 for the volume of gel required. For example, 100 ml of a 1.5% gel is made by weighing out 1.5 gram of TreviGel 500 powder. 2. Prepare enough 1X TAE buffer for both casting and running of the gel. Add the 1X TAE buffer to a clean Erlenmeyer flask (100 ml in this example). Transfer the TreviGel powder to the Erlenmeyer flask and swirl to make certain that the gel is in suspension. Place the flask on a top loading balance, and tare or record the weight. 3. Microwave on medium to medium-high setting until no particulates are visible in the molten gel. The time will vary, since microwave units vary in power. In general, TreviGel 500 requires heating for about one minute longer than agarose. Gently swirl flask to mix. HANDLE WITH CARE, the molten TreviGel 500 is very hot. Replace water that evaporated during microwaving by returning to top loading balance and adding distilled water until the tared weight (or recorded weight) is reached. Gently swirl to mix. 4. Once bubbles have stopped forming, wait an additional minute, and pour the gel into a casting tray. Allow the gel to cool for 30 minutes. 5. For optimal results, place gel at 4 C for 30 minutes before electrophoresing samples 6. To each labeled DNA sample (10 µl), add 2 µl of 5X gel loading dye. Load samples into wells and electrophorese at 50 to 100 V for about 2 hours. Conditions may vary with electrophoresis setup used. -7-

8 IX. Trouble Shooting Guide PROBLEM POSSIBLE CAUSE RECOMMENDATIONS DNA recovery Inadequate lysis of sample Mix equal volumes of Lysis solution 1 with sample in Sample buffer. Phase Inversion Inadequate level of labeling Lysis solution 1 not diluted sufficiently Lysate too viscous Carryover of organic phase Klenow enzyme may be inactive Make sure to add equal volumes of Lysis solution 1 with the sample in Sample buffer. Resuspend the sample in a larger volume of Sample buffer and Lysis solution 1. Be careful during organic separations to avoid transfer of the organic phases, which may inhibit the enzymatic reaction. A chloroform extraction can be helpful to remove some of these organics. If the Klenow enzyme is old (greater than one year old) or has been subjected to multiple freeze thaws or has been improperly stored (e.g. in a frostfree freezer) then it may become inactive. Note: It is alway best not to thaw any enzyme to ice temperature. Remove the volume of enzyme required, while the enzyme is at -20 C and return it immediately to the freezer, without a warm up stage on ice. Film overexposed or black All samples show little or no apoptotic DNA fragmentation All samples, including the negative control show extensive DNA degradation (Appears as a smear) Inadequate washing after streptavin HRP binding Non-fat milk contains biotin Nylon membrane is charged Tissue culture samples may not be undergoing apoptosis. Kit used not sensitive enough Harsh treatment of DNA Negative control is inappropriate Increase number and duration of washes following Strep-HRP incubation. Switch brand or try 1-2 %BSA. Swith to uncharged nylon membrane. Conditions for inducing apoptosis may need to be changed. These conditions will vary with the cell type. Try Isotopic kit (highest sensitivity) If the DNA is isolated in a very vigorous manner, the DNA may become sheared. The DNA should be isolated again from fresh samples, but under less aggressive isolations procedures. The negative control may be undergoing apoptosis and may therefore be inappropriate as a control. The choice of an alternative negative control may be warranted. Unusual banding pattern RNA present in sample. Some cell types contain high levels of RNA which may appear when using the ethidium bromide kit. Usually 2 bands are present. RNA may be removed by adding 20 µg ml -1 RNase A (DNase free!) to the Sample Buffer, or to the resuspended DNA. Also, gels may be soaked in 1-5 µg/ml RNase A in water for 30 minutes at room temperature with shaking. Other questions?, Contact Trevigen Technical Support at TREVIGEN or at info@trevigen.com -8-

9 X. Appendix Buffer Composition: 50X TAE Buffer (Cat. # ): 2.0 M Tris, ph M Sodium Acetate 50 mm EDTA 10X Klenow Buffer (Cat. # ): 100 mm Tris, ph mm MgCl 2 20X SSC: 3 M NaCl 0.3 M NaCitrate, ph X PBS (Cat. # ): 75 mm Na 2 HPO 2 25 mm NaH 2 PO M NaCl DL dntp Mix (Cat. # ): 7.5 mm datp 7.5 mm dgtp 7.5 mm dttp 0.25 mm biotinylated dctp Warning and Precautions: The DNA extraction reagents contain ingredients that may be irritating or caustic if allowed to come in contact with skin, eyes or mucous membranes. Extraction solution 2 contains combustible ingredients, therefore keep it away from excessive heat, sparks and flame. Do not mix this reagent with strong oxidizing agents. -9-

TACS TM DNA Laddering Kit

TACS TM DNA Laddering Kit Apoptosis Detection Kit catalog number: TA4632 Chemiluminescent 20 tests FOR RESEARCH USE ONLY NOT FOR USE IN DIAGNOSTIC PROCEDURES. Contents TABLE OF CONTENTS Page PRINCIPLE