EXPERIMENT 26: Detection of DNA-binding Proteins using an Electrophoretic Mobility Shift Assay Gel shift Remember to use sterile conditions (tips, tubes, etc.) throughout this experiment Day 1: Biotinylation Reaction 1. Keep all components and reactions on ice until tubes are ready for the reaction incubation. TdT will be kept at -20 in a bench top cooler at the TA bench 2. Collect a nuclease-free microfuge tube containing either the SP1 or AP2 consensus oligonucleotide. Be sure to write which one you have obtained in your lab notebook. 3. Assemble the following reaction in order in a sterile nuclease-free microfuge tube. Label the tube as SP1 or AP2. Gently mix the extracts with a pipette tip upon the addition of each component. Be sure to use the P2 pipettor provided for volumes of 2 µl or less. Component Volume (µl) Ultrapure nuclease-free water 25 5x TdT Reaction Buffer 10 Final concentration N/A Unlabeled oligo (1 µm) 5 100 nm Biotin-11-UTP (5 µm) 5 Diluted TdT (2U/µl) 5 1x 0.5 µm 0.2 U/µl 10. Incubate the reaction in a 37 C water bath for 30 min. Be sure to replace the cover after placing the tubes in the water bath. 11. Stop the reaction by adding 2.5 µl of 0.2 M EDTA. 12. In the hood, add 50 µl of chloroform:isoamyl alcohol (24:1) to the reaction to extract the TdT. Vortex the mixture briefly 13. Centrifuge the mixture for 2 minutes at maximum speed to separate the phases. 14. Transfer the aqueous (top) phase to a new, labeled, sterile microfuge tube, keep on ice. 15. Turn in your remaining, unlabeled DNA for storage for Day 2. Label the tube with your group initials. Determination of Labeling Efficiency Do not touch the nylon membrane with your skin or with contaminated gloves 1. Using a pencil, lightly draw on a piece of 7 cm x 9 cm nylon N+ membrane a slanted mark in the upper left corner (to indicate orientation ) and a 6 x 8 grid with a 0.5 cm surrounding zone as follows. Label the edges of a grid as your 96-well plate is labeled. See below: 1
0.5 cm 0.5 cm 0.5 cm 1.3 cm 1.3 cm 1.3 cm 1.3 cm 1.3 cm 1.3 cm A 1 2 3 4 5 6 B C D E F G H 0.5 cm 2. Hydrate your nylon N+ membrane in a clean tray with 10 ml TE buffer for 10 15 minutes. 3. Obtain control biotinylated DNA and control unlabeled DNA that has been diluted with TE to a final concentration of 20 nm. 4. Dilute a portion of your test sample (either SP1 or AP2) in TE to a final concentration of 10 nm by combining 6 µl of your reaction with 54 µl TE. 5. Create standard % Biotin dilutions: Tube % Biotinylated DNA Biotinylated control DNA (µl) Unlabeled control DNA (µl) TE buffer (µl) Biotinylated DNA (fmol) 1 100 12 0 48 25 2 75 9 3 48 18.75 3 50 6 6 48 12.5 4 25 3 9 48 6.25 5 0 0 12 48 0 6. Pipette 25 µl of TE into wells 1-6 in rows B-H of a low protein absorption 96-well plate (do not pipette TE into wells A1-6). 7. Pipette 50 µl of samples 1-5 into wells A1-5 and 50 µl of your test (either AP2 or SP1) sample into well A6. 2
8. Remove 25 µl from each well in row A and add to the corresponding well in row B. Mix by pipetting up and down. 9. Remove 25 µl from each well in row B and add to the corresponding well in row B. Mix by pipetting up and down. 10. Repeat step 8 for the remaining rows. 11. Remove the nylon membrane from the TE buffer and blot on paper towel. The membrane should be moist but not wet. Do not allow the membrane to dry out, but ensure that there are no puddles of buffer on the membrane. 12. Spot 2 µl of each sample into the corresponding position on your membrane. It is recommended that you do not plunge all the way to the second stop with your P2 pipette. 13. To crosslink the DNA to the membrane, cover the surface of the transilluminator with fresh clear plastic wrap; place your blot, face-down, on a UV transilluminator. The transilluminators are equipped with 300-nm wavelength bulbs. Turn on the transilluminator and expose your blot for 10 minutes. Remember to protect your eyes and skin from the UV by wearing gloves, goggles and/or a facemask. Throw away your used clear plastic wrap. Imaging your blot 1. Block membrane in 16 ml of warmed blocking buffer in a clean container for 15 minutes with gentle shaking. 2. Combine 50 µl of Streptavidin-HRP conjugate with 16 ml of warmed blocking buffer and mix by inverting. 3. Pour blocking buffer off of blot (the blot will likely stick to the bottom of the container, avoid touching the blot). Add the diluted Streptavidin-HRP conjugate in blocking buffer solution and incubate for 15 minutes with gentle shaking. 4. Obtain 40 ml of warmed 4x wash solution. Dilute this to 1x by combining it with 120 ml H 2 O. 5. Transfer the membrane to a clean container. Rinse the membrane once with 20 ml of 1x wash buffer. 6. Wash the membrane 4 additional times with ~30 ml of 1x wash buffer for 5 minutes each with gentle shaking. 7. Transfer the membrane to a clean container. Incubate with 30 ml of substrate equilibrium buffer for 5 minutes with gentle shaking. 8. Prepare the chemiluminescent substrate working solution by combining 6 ml of enhancer solution and 6 ml of stable peroxide solution and mix by inverting or vortexing. Note that this is a lightsensitive procedure from this step forward. Avoid prolonged exposure of this solution and the blot post-treatment with this solution with light. 9. Pour a puddle of the working solution into a clean container. Remove the membrane from the equilibrium buffer and blot gently on a paper towel. Do not allow the blot to become dry. Place the blot, face-down, on the working solution puddle. 3
10. Incubate the blot on the working solution for 5 minutes without shaking. 11. Remove the blot from the working solution and blot gently on a paper towel. Do not allow the blot to become dry. Slide the blot into a labeled plastic zip-top bag, press out all of the air and seal tightly. 12. The blot will be exposed and developed by the TA. Analysis of efficiency of biotinylation 1. Your film will be available electronically on the course webpage. You will use ImageJ to calculate the intensity of the dots on your blot (note that these directions are for use on a PC). Open ImageJ. Click file, open and select your file. 2. Subtract the background by clicking process, subtract background. Select a rolling ball radius of 25 50 taking care not to wash out the most intense dots. Invert the colors by selecting edit, invert. Enable Integrated Density by selecting analyze, select measurements, integrated density. 3. Select the circle tool and draw a circle around the first dot. It is important that the circle be larger than the dot to be analyzed and to use the same exact circle on every dot, so ensure that the circle will fit around all of the dots that you will be analyzing. Select analyze, measure for a reading. Repeat for all remaining dots. It is recommended that you work across each row of controls and include the 0% biotinylation (the value will be near 0). 4. Create a scattered line plot of the data by plotting the IntDen versus the % Biotin. Create a linear trendline and use this line equation to determine the efficiency of your biotinylation. Non-linear data points can result from saturation of pixels at a specific dot. It might be necessary to remove the most intense dot from your calculations if it is not linear. It would be incorrect to remove the 0% Biotin data points from data. Where any data points removed from your calculations? Why? What percent biotin is present in your sample? How many fmol are present? Day 2: DNA Binding Reactions 1. Obtain a 10-well, BioRad 5% TBE acrylamide gel. Place the gel in the electrophoresis apparatus; each tank holds 2 gels (2 groups will share one tank). Fill the inside of the gel area and the bottom of the tank with 0.5x TBE. Each tank will require approximately 400 ml of 0.5% TBE. 2. Flush out the wells with 0.5x TBE that is present in the tank. Pre-run the gel for 30-60 minutes at 100 V. 3. Assemble the following reactions, in order, in sterile nuclease-free 0.5-ml labeled microfuge tubes. Each group will assemble all of the reactions listed in the table below. NOTE: groups that use the labeled oligo for AP2 will use unlabeled AP2 oligo as the specific competitor and groups that use the labeled oligo for SP1 will use unlabeled SP1 oligo as the specific competitor. 4
The control reaction Component Reaction #1 Reaction #2 Reaction #3 Ultrapure water 12 µl 7 µl 3 µl 10x Binding Buffer 2 µl 2 µl 2 µl 50% Glycerol 1 µl 1 µl 1 µl 100 mm MgCl 2 1 µl 1 µl 1 µl 1 µg/µl Poly(dI dc) 1 µl 1 µl 1 µl 1% NP-40 1 µl 1 µl 1 µl Biotinylated EBNA Oligo 2 µl 2 µl 2 µl Specific Competitor EBNA Oligo -- -- 4 µl EBNA Nuclear Extract -- 5 µl 5 µl And the test reaction Component Reaction #4 Reaction #5 Reaction #6 Ultrapure water 12 µl 7 µl 3 µl 10x Binding Buffer 2 µl 2 µl 2 µl 50% Glycerol 1 µl 1 µl 1 µl 100 mm MgCl 2 1 µl 1 µl 1 µl 1 µg/µl Poly(dI dc) 1 µl 1 µl 1 µl 1% NP-40 1 µl 1 µl 1 µl Biotinylated Test Oligo (either AP2 or SP1) Specific Test Competitor Oligo (either AP2 or SP1) 2 µl 2 µl 2 µl -- -- 4 µl HeLa Nuclear Extract -- 5 µl 5 µl 4. Mix by gently pipetting up and down then quickly spin down. Incubate at room temperature for 20 minutes. 5. Add 5 µl 5x loading buffer to each sample and mix by GENTLY pipetting up and down. (Only pipette up and down the minimal amount of times, once or twice, to mix the solution. Repeated pipetting could disrupt protein:dna interactions.) 6. Stop the pre-running gel and flush the wells once more with 0.5x TBE. Using a gel loading tip, load 15 µl of each sample, as follows: 5
Lane 1: empty Lane 2: reaction #1 Lane 3: reaction #2 Lane 4: reaction #3 Lane 5: empty Lane 6: reaction #4 Lane 7: reaction #5 Lane 8: reaction #6 Lane 9: empty Lane 10: empty 7. Label a piece of tape with the initials of both groups and place it on the outside of the tank on the side containing the designated gel. Note that both groups that are sharing a single tank must be ready to load the gel before any samples are loaded. This prevents the samples from floating out of the wells before the current is applied. 8. Run the gel at 100 V until the bromophenol blue (bottom) dye front is about 3/4 of the way through the gel. Transferring the blot Four gels will share a semi-dry blot transfer system for this portion of the lab. 1. Obtain one 9 cm x 7 cm nylon membrane per gel and 6 pieces of 3mm thick Whatman paper per gel. 2. Initial the top, left-hand corner of the membrane with pencil. Soak the nylon membrane in 0.5x TBE for at least 10 minutes. Wet the six sheets of Whatman paper (separately) just before use in the same buffer. 3. Stack the first three wet pieces of Whatman paper on the transfer apparatus (See Fig 1). 4. Place the nylon membrane, face up on top of the Whatman paper. 5. Open up the gel plates and gently place the gel on top of the nylon membrane. Orient the wells with well 1 in the top left-hand corner (next to group one s initials) and well 15 in the top left hand corner. Ensure that the line down the middle of the membrane aligns with lane 8 of the gel. 6. Lay the last three pieces of wet piece of Whatman paper on top of the gel. 6
7. When all of the groups have assembled their transfers, fasten the lids of the transfer apparatus. Do not reverse the electrodes. Run the transfer at a constant voltage for 30 minutes. Gradually increase the voltage during the run (without turning off the power supply). Begin with 5 minutes of 5 V, the do 10 minutes of 10 V, 10 minutes of 15 volts and 5 minutes of 20 volts. 8. When finished, look for the dye fronts on the membrane. If any dye remains in the gel, see the TA to finish transferring. Rinse the bottom of the transfer apparatus and the lid with tap water, then rinse with deionized water. 9. To crosslink the DNA to the membrane, cover the surface of the transilluminator with fresh clear plastic wrap; place your blot, face-down, on a UV transilluminator. The transilluminators are equipped with 300-nm wavelength bulbs. Turn on the transilluminator and expose your blot for 10 minutes. Remember to protect your eyes and skin from the UV by wearing gloves, goggles and/or a facemask. Throw away your used clear plastic wrap. 10. Place your blot, face up in the designated hood for storage. Day 3: Imaging your blot 1. Block membrane in 16 ml of warmed blocking buffer in a clean container for 15 minutes with gentle shaking. 2. Combine 50 µl of Streptavidin-HRP conjugate with 16 ml of warmed blocking buffer and mix by inverting. 3. Pour blocking buffer off of blot (the blot will likely stick to the bottom of the container, avoid touching the blot). Add the diluted Streptavidin-HRP conjugate in blocking buffer solution and incubate for 15 minutes with gentle shaking. 4. Obtain 40 ml of warmed 4x wash solution. Dilute this to 1x by combining it with 120 ml H 2 O. 5. Transfer the membrane to a clean container. Rinse the membrane once with 20 ml of 1x wash buffer. 6. Wash the membrane 4 additional times with ~30 ml of 1x wash buffer for 5 minutes each with gentle shaking. 7. Transfer the membrane to a clean container. Incubate with 30 ml of substrate equilibrium buffer for 5 minutes with gentle shaking. 8. Prepare the chemiluminescent substrate working solution by combining 6 ml of enhancer solution and 6 ml of stable peroxide solution and mix by inverting or vortexing. Note that this is a lightsensitive procedure from this step forward. Avoid prolonged exposure of this solution and the blot post-treatment with this solution with light. 9. Pour a puddle of the working solution into a clean container. Remove the membrane from the equilibrium buffer and blot gently on a paper towel. Do not allow the blot to become dry. Place the blot, face-down, on the working solution puddle. 10. Incubate the blot on the working solution for 5 minutes without shaking. 7
11. Remove the blot from the working solution and blot gently on a paper towel. Do not allow the blot to become dry. Slide the blot into a labeled plastic zip-top bag, press out all of the air and seal tightly. 12. The blot will be exposed and developed by the TA. The data file will be available on the course webpage. 11. Explain the results for each of your six reactions. How many bands are present in each lane and what molecule or complex is represented by each of these bands? Do your results agree with what you expected to see? If more than one band is present, why are there two species instead of one? Was the complex seen in your positive control reaction specific? What was the reaction efficiency of your biotinylation experiment? Do you think that the efficiency effected reactions 4 6? Explain your conclusions. 8