Biomod/2014/OhioMOD/protocols: Difference between revisions

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<div id="protocols"> <h1>Lab Protocols</h1>

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     <TH COLSPAN="2"><strong>Table of Contents</strong>
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<A HREF="#scroll1">1. DNA origami structure production </br><ul id="list2"><A HREF="#scroll1.1">1.1. Pre-stocks</ul> <ul id="list2"><A HREF="#scroll1.2">1.2. Working stocks</ul> <ul id="list2"><A HREF="#scroll1.3">1.3. Folding reactions</ul> <A HREF="#scroll2">2. Imaging <ul id="list2"><A HREF="#scroll2.1">2.1. Agarose gel electrophoresis <ul id="list2"><A HREF="#scroll2.1.1">2.1.1. UV imaging</ul> <ul id="list2"><A HREF="#scroll2.1.2">2.1.2. Typhoon imaging</ul></ul> <ul id="list2"><A HREF="#scroll2.2">2.2. Sample preparation for TEM</ul> <ul id="list2"><A HREF="#scroll2.3">2.3. Sample preparation for TIRF</ul> <A HREF="#scroll3">3. Purification </br><ul id="list2"><A HREF="#scroll3.1">3.1. Agarose gel electrophoresis</ul> <ul id="list2"><A HREF="#scroll3.2">3.2. PEG purification</ul> <A HREF="#scroll4">4. Fluorescence <ul id="list2"><A HREF="#scroll4.1">4.1. TOPRO3 intercalating dye</ul> <ul id="list2"><A HREF="#scroll4.2">4.2. Lysotracker Green dye</ul> <A HREF="#scroll5">5. Cells <ul id="list2"><A HREF="#scroll5.1">5.1. Cell splitting</ul> <ul id="list2"><A HREF="#scroll5.2">5.2. Cell counting</ul> <A HREF="#scroll6">6. Gene Expression <ul id="list2"><A HREF="#scroll6.1">6.1. RNA isolation</ul> <ul id="list2"><A HREF="#scroll6.2">6.2. qPCR</ul> <ul id="list2"><A HREF="#scroll6.3">6.3. Western Blot<A NAME="scroll1"></A>.</ul> </TD>

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<div id=1> <h2><A NAME="scroll1.1"></A>1. DNA origami structure production</h2> <h3><u>1.1 Pre-Stocks</u></h1> The oligonucleotide staples required for DNA origami structure production generally come in 96 well plates, with a single oligonucleotide in each well.

</br></br>The purpose of creating pre-stocks is to segregate all staples necessary to assemble a particular section of the structure into one stock, to make folding reaction preparation more convenient in the future.

</br></br>To create pre-stocks: <ul id="list2">1. Determine which staples can be combined into one stock, and <ul id="list2">allocate wells for each stock in a 96-well plate.</ul> </ul><ul id="list2">2. Centrifuge the oligonucleotide plates at 2000 rpm for 2 minutes. </ul><ul id="list2">3. Pipet the desired oligonucleotides into the stock well. <ul id="list2">a. For 1-2 oligos, use 20 uL.</ul> <ul id="list2">b. For 2-10 oligos, use 15 uL.</ul> <ul id="list2">c. For >10 oligos, use 10 uL.</ul> <ul id="list2">d. For >30 oligos, use a trough and a multichannel pipet to mix <ul id="list2">different oligos before transferring into well.</ul></ul></ul></ul> <ul id="list2"><A NAME="scroll1.2"></A>4. Mix the contents in each pre-stock well thoroughly by pipetting.</ul>

<h3><u>1.2 Working stocks</u></h1> A working stock is the combination of all the oligonucleotide staples in the correct proportion necessary to fold a DNA origami structure.

</br></br>To create working stocks: <ul id="list2">1. Determine the volume of oligos required to fold structure properly.</ul> <ul id="list2">2. Centrifuge pre-stocks plate to bring down any liquid on the sides or<ul id="list2"> the top.</ul></ul> <ul id="list2">3. In a 1.5 mL tube, add the correct amount of pre-stock oligonucleotides <ul id="list2">previously determined.</ul></ul> <ul id="list2">4. Vortex solution for ~5 sec.</ul> <ul id="list2"><A NAME="scroll1.3"></A>5. Centrifuge for ~10-15 sec.</ul>

<h3><u>1.3 Folding reactions</u></h1> <ul id="list2">1. Prepare working stock.</ul> <ul id="list2">2. For a 20 nM scaffold concentration in 50 uL volume with 10x staple <ul id="list2">concentration:</ul> <ul id="list2">a. Add 10 uL of 100 nM scaffold.</ul> <ul id="list2">b. Add 20 uL of 500 nM staples.</ul> <ul id="list2">c. Add 10 uL of dH2O.</ul> <ul id="list2">d. Add 5 uL of 10x FOB buffer.</ul> <ul id="list2">e. Add 5 uL of MgCl2 salt in desired concentration.</ul></ul> <ul id="list2">3. Mix by pipetting.</ul> <ul id="list2"><A NAME="scroll2"></A>4. Place in thermocycler with desired temperature cycles and time ramp.</ul> </div>

<div id=2> <h2><A NAME="scroll2.1"></A>2. Imaging</h2> <h3><u>2.1. Agarose gel electrophoresis</u></h1> For 2% Agarose gels:

</br></br><u>Large gel:</u> </br>1. Measure out 2.5g Agarose in beaker. </br>2. Add 124g 0.5x TBE buffer. </br>3. Microwave for 1-2 mins to dissolve Agarose. </br>4. Replace evaporated water with dH2O until back to 124g. </br>5. Add 1 mL of 1.375 M MgCl2. </br>6. Add 5 uL ethidium bromide (EtBr) intercalating dye.* </br>7. Swirl to mix, then add into gel tray and insert comb.

</br></br><u>Small gel:</u> </br>1. Measure out 1g Agarose in beaker. </br>2. Add 49.6g 0.5x TBE buffer. </br>3. Microwave for 1-2 mins to dissolve Agarose. </br>4. Replace evaporated water with dH2O until back to 49.6g. </br>5. Add 0.4 mL of 1.375 M MgCl2. </br>6. Add 2 uL ethidium bromide (EtBr) intercalating dye.* </br>7. Swirl to mix, then add into gel tray and insert comb.

</br></br>*Add EtBr only if imaging using UV light.

</br></br><u>Gel loading:</u> </br>1. Mix 15 uL of sample with 3 uL of 6x loading dye. <ul id="list2">a. For scaffold at 100 nM, mix 1.5 uL scaffold with 13.5 uL dH2O and 3 uL <ul id="list2">dye.</ul></ul> 2. Once gel is solid, fill gel rig with TBE buffer containing MgCl2. </br>3. Remove comb. </br>4. Add 17 uL of prepared samples into the wells in the gel. <ul id="list2">a. For DNA ladder, add 6 uL.</ul> <A NAME="scroll2.1.1"></A>5. Attach electrodes, connect to power supply, and run at 70 volts. <ul id="list2">a. Small gels can be run for ~2 hours, large gels may require more than 4 <ul id="list2">hours.</ul></ul>

<h4><u>2.1.1 UV imaging:</u></h4> 1. Ensure that the gel contains EtBr. </br>2. Place gel on a UV imaging table. <ul id="list2">a. The EtBr should make DNA fluoresce under UV radiation.</ul> 3. Use a camera to image the gel. <ul id="list2"><A NAME="scroll2.1.2"></A>a. Change the brightness, contrast, or focus to ensure that bands show <ul id="list2">up clearly in image.</ul></ul>

<h4><u>2.1.2 Typhoon imaging:</u></h4> 1. Ensure that the gel DOES NOT contain EtBr. </br>2. Determine the wavelength at which fluorescent markers are excited. </br>3. Place gel in a Typhoon 9410 imager. </br>4. Select the signal type, excitation wavelength, orientation and focal height. </br>5. <A NAME="scroll2.2"></A>Image gel.

<h3><u>2.2. Sample preparation for TEM</u></h1>

<u>UFO preparation</u> </br>1. Add 5 mL of boiling deoxygenated ddH2O to 0.1g UFO powder. </br>2. Vortex vigorously for 10 mins. The solution should be light brown. </br>3. Filter solution. It should now be clear. </br>4. Aliquot 200 uL into separate tubes. </br>5. Centrifuge tube at max speed for 5 mins on a tabletop centrifuge. </br>6. Add 1 uL of 5 M NaOH to the side of the stain solution tube. Do not add it <ul id="list3">directly into the solution.</ul> 7. Vortex immediately for 2 mins. </br>8. Centrifuge at top speed for 3 mins. </br>9. Wrap tube with foil to keep away from light

</br></br><u>UFO staining</u> </br>1. Glow-discharge the carbon-coated TEM grids to make surface hydrophilic. </br>2. Apply ~3 uL of sample solution onto carbon-coated side of grid. Let it adsorb<ul id="list3"> into the grid for 4 mins.</ul> 3. After 4 mins, use filter paper to draw off liquid from the edge of the grid. Do <ul id="list3">not touch the grid surface.</ul> 4. Immerse grid sample-side first into a droplet of 10 uL 2% UFO staining <ul id="list3">solution. Remove stain solution immediately using a filter paper.</ul> 5. Immerse grid sample-side first into a droplet of 20 uL UFO. Let the grid face <ul id="list3">soak in the staining solution for 40 sec. Afterward, remove the solution using a filter paper.</ul> <A NAME="scroll2.3"></A>6. Let the grid dry for at least 15 mins before inserting into the TEM.

<h3><u>2.3. Sample preparation for TIRF</u></h1> <u>TIRF 8-well cell plate preparation</u> </br>1. Remove ~2 mL of cell suspension from stock suspension. <ul id="list2">a. equalize volume in stock with warm (37 C) human RPMI</ul> 2. Count cells in cell suspension (see protocol). </br>3. Aliquot 1x105 cells into an epi tube. </br>4. Wash cells in PBS twice. <ul id="list2">a. Centrifuge cells at 300 G for 5 mins. <ul id="list2">i. When putting tube in the centrifuge, ensure that the hinge of the <ul id="list2">cap is facing outwards, so that cell pellet will always form by the<ul id="list2">hinge.</ul></ul></ul></ul> <ul id="list2">b. Aspirate out as much as the supernatant as possible without sucking<ul id="list2"> up pellet.</ul></ul> <ul id="list2">c. Resuspend cell pellet by adding 500 uL of PBS. <ul id="list2">i. Dispense liquid by the rim of the tube.</ul></ul> <ul id="list2">d. Vortex for <1 sec to mix in pellet.</ul> <ul id="list2">e. Centrifuge at 300 G for 5 mins to form pellet again.</ul> <ul id="list2">f. Aspirate to remove supernatant.</ul> 5. While washing cells, add poly-L-lysine onto a circular plate until the surface<ul id="list3"> of the inner circle is covered.</ul> 6. Let the poly-L-lysine incubate for 5 minutes, and then wash twice with PBS<ul id="list3"> solution.</ul> <ul id="list2">a. To wash, remove the excess poly-L-lysine, add similar amount of PBS <ul id="list3"> on the plate, and remove after a few seconds.</ul></ul> 7. Stain washed cells with fluorescent dye of choice according to protocol. </br>8. Centrifuge at 300 G for 5 mins, and remove supernatant. </br>9. Wash cells twice in clear media and resuspend in same media. </br>10. Pipet ~400 uL of cell suspension onto the plate. </br>11. Add PBS to surrounding cells to prevent evaporation of sample.

</br></br><u>TIRF microscope's lide preparation</u> </br>1. Take a microscope slide, and place it at a slight incline. </br>2. Place 2 strips of double sided tape to form a channel in between the 2 pieces <ul id="list3">of tape.</ul> 3. Place a cleaned coverslip on the pieces of tape. </br>4. Press down on the tape with a small epi tube to seal the channel on the <ul id="list2">sides.</ul> 5. Pipet ~20 uL of dilute strep into the channel. <ul id="list2">a. Place the tip of the pipet by the opening between slide and coverslip and pipet slowly. The liquid should move into the channel by capillary action, helped along by the incline. Once the whole channel is full, stop pipetting.</ul> 6. Let the strep incubate for 5 minutes. </br>7. Wash excess strep away using PBS buffer. <ul id="list2">a. Pipet in PBS from the top and hold a piece of filter paper at the bottom to wick away the excess liquid being displaced on the other end. Try to match the speed of pipetting to the speed of absorption by the filter paper to prevent formation of bubbles. If a bubble blocks flow on one end, pipet from the other end to force bubble out. Small bubbles don’t really matter.</ul> 8. Pipet ~20 uL of BSA/casein into the channel. <ul id="list2">a. Same procedure as previous step.</ul> 9. Let the BSA/casein incubate for 5 minutes. </br>10. Wash with PBS buffer. </br>11. Pipet ~20 uL of structures. </br>12. Incubate for 5 minutes. </br>13. Wash with PBS buffer. </br><A NAME="scroll3"></A>14. Seal off the ends of the channel.

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<div id=3> <h2><A NAME="scroll3.1"></A>3. Purification</h2> <h3><u>3.1. Agarose gel electrophoresis</u></h1> For gel preparation, please see <A HREF="#scroll2.1">section 2.1.</A> </br></br><u>Gel purification:</u> </br>1. Ensure that gel contains EtBr. </br>2. Place gel on UV imaging table. </br>3. Cut out gel pieces that display DNA fluorescence and may correspond to <ul id="list3">structures.</ul> 4. Place gel pieces into the filter of a freeze n’ squeeze tube. </br>5. Centrifuge at 13,000 g for 6 mins. </br><A NAME="scroll3.2"></A>6. Remove filter and store liquid containing purified structures.

<h3><u>3.2. PEG purification</u></h1> 1. In a 1.5 mL tube, add the desired volume of structure. </br>2. Add the same volume of 15% PEG 8000. Vortex well to mix. </br>3. Centrifuge at 16,000 g for 30 mins. </br><A NAME="scroll4"></A>4. Resuspend in buffer of choice.

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<div id=4> <h2><A NAME="scroll4.1"></A>4. Fluorescence</h2> <h3><u>4.1. TOPRO3 intercalating dye</u></h1> 1. Calculate the concentration of structure in desired volume. </br>2. Add correct volume of intercalating dye into structure solution. </br>3. For 1 TOPRO3 molecule every 10 base pairs from a stock of 1 mM dye: </br></br> (volume of origami (μL) × concentration origami (nM) × length of scaffold(bp) ×〖 10〗^(-7))

</br>= TOPRO3 (μL) </br></br>1. Combine desired amount of origami with calculated TOPRO3. </br>2. Let sit for 2-24 hours: can speed up process by placing in shaker/incubator at <ul id="list3">37oC and shake. </ul> 3. Centrifuge at 16,000 xg+ for 25 minutes. Should have visible pellet. </br>4. Remove supernatant/ discard </br><A NAME="scroll4.2"></A>5. Resuspend pellet with desired buffer (same volume as before)

<h3><u>4.2. Lysotracker Green dye</u></h1>

1. Count Cells </br>2. Get volume for 500,000 cells. </br>3. Wash twice with PBS. </br>4. Add lysotracker dye to media: <ul id="list2">a. Concentration of bottle of lysotracker = 1 mM</ul> <ul id="list2">b. Final concentration in media = 100 nM</ul> 5. Warm media+dye to 37 degrees </br>6. Add warm media to cells </br>7. Let cells incubate in dye+media for 1.5 hours </br><A NAME="scroll5"></A>8. Centrifuge and resuspend cells in clear media before imaging

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<div id=5> <h2><A NAME="scroll5.1"></A>5. Cells</h2> <h3><u>5.1. Cell splitting</u></h1> 1. Pipet ~10 mL of Human RPMI from the stock solution into a falcon tube <ul id="list3">using a serological pipet and let it warm up to 37 C in the incubator.</ul> 2. Take the cells out of the refrigerator and observe them under the <ul id="list3">microscope to see how the cells are doing.</ul> 3. Remove 8 mL of the cell media from the flask. <ul id="list2">a. Make sure to mix vigorously before removing the media.</ul> <ul id="list2">b. The ideal way to do this is to suck up all the media in the flask, then deposit 2 mL back into the flask. This way, we are always sure of the amount of old media in the flask.</ul> 4. Pipes 8 mL of the warm human RPMI into the flask, and pipet a few times to <ul id="list3">ensure proper mixing.</ul> <A NAME="scroll5.2"></A>5. Store the flask back into the incubator.

<h3><u>5.2. Cell counting</u></h1>

1. Transfer 10 mL of cells into a 15 mL falcon tube. </br>2. Add 10 uL of dye (Trypan - Blue) to an eppendorf tube </br>3. Add 10 uL of cells to the same tube. <ul id="list2">a. Should be a 1:1 ratio of cells and dye</ul> 4. Add 10 uL of the mixture to the hemocytometer under the cover slip. </br>5. Put the hemocytometer on the scope, turn the scope on, focus image. </br>6. Count the cells in the 4 corners of the cover slip. </br>7. Calculate the number of cells: (total from 4 corners)/4*2*10,000 = total <ul id="list3">cells/mL</ul> 8. Wipe coverslip and hemocytometer with ethanol<A NAME="scroll6"></A>. </div>

<div id=6> <h2><A NAME="scroll6.1"></A>6. Gene Expression</h2> <h3><u>6.1. RNA isolation</u></h3> </br><i>This protocol is optimized for 3 x 105 to 1 x 1077 cells.</i> </br></br>1. Wash cells twice with PBS. </br>2. After second wash, remove supernatant and add 0.5 to 1 mL of Trizol reagent. Vortex for ~2 minutes. </br>3. Incubate at room temperature for 5 minutes. </br>4. Add 200 uL of chloroform. Vortex briefly. </br>5. Incubate at room temperature for 3 minutes. </br>6. Centrifuge at 12,000 g for 15 minutes. </br><ul id="list2"> After centrifugation, RNA will be on the top aqueous phase, DNA will be in the buffy coat, and rest of cell lysate will be in pink Trizol phase.</ul> 7. Transfer clear aqueous phase into a separate tube. Add 500 uL of 100% isopropanol. 8. Incubate at room temperature for 10 minutes. Centrifuge at 12,000 g for 10 minutes. </br>9. Remove supernatant, and resuspend in 75% ethanol. Centrifuge at 7,500 g for 5 minutes. </br>10. Remove supernatant. Let pellet air dry for 5-60 minutes. </br>11. Resuspend in 20 to 40 uL of dH2O. </br>12. Measure concentration of RNA by measuring absorbance of solution at 260 nm wavelength. </br><ul id="list2"> Determine purity by calculating the ratio between the 260 nm and the 280 nm readings.</ul> 13. Add 2.5 ug of extracted RNA to 2.5 uL of 10x DNAse buffer and 2.5 uL of DNAse I (amplification grade). </br>14. Bring volume up to 25 uL using DNAse/RNAse free water. </br>15. Incubate at room temperature for 15 minutes. </br>16. Add 2 uL of 25 mM EDTA. Incubate at 65 C for 10 minutes. </br><ul id="list2"> This will heat inactivate the DNAse I.</ul>

<strong>Reverse transcription (RT) to cDNA</strong> </br>1. Prepare master mix in provided ratios: </br><a><img src="http://openwetware.org/images/2/20/Prottable.png"height="200" width="500"/></a> </br>2. Add 14 uL of DNAse treated RNA to 6 uL of master mix. </br>3. Incubate at 37 C for 1 hour.

<h3><u>6.2. qPCR</u></h3> </br><u>Sample Prep</u> </br></br>1. Thaw one vial of “Reaction Mix” and “Enzyme” and shield it from light. </br><ul id="list2">a. If precipitate is observed inside vial, place Reaction Mix in shake ‘n bake at 37 C until precipitate has dissolved.</ul> 2. Centrifuge both vials briefly and place on ice. </br>3. Pipette 10 uL of “Enzyme” into “Reaction Mix” tube. Mix gently by pipetting. </br>4. Store on ice until ready to use, use within one week. </br>5. In a 1.5 mL epi tube, prepare PCR Mix by adding the following components. </br><a><img src="http://openwetware.org/images/6/6e/Tableqpcrprot.png"height="200" width="500"/></a> </br>6. Mix gently by pipetting. Add 18 uL of reaction mix to PCR plate. Add 2 uL of template(specific scaffold) DNA last, and cover the receptacle. a. For standard curves, use serial dilutions of target gene at known concentrations. b. As negative control, replace template DNA with PCR grade water. 7. Centrifuge solution at 700-1000g for 5-10 sec. </br>8. Place receptacle into qPCR machine and start. </br></br><u>qPCR instrument setup.</u> </br>1. Pre-incubation step: 10 min at 95 C to activate enzyme and denature DNA. </br>2. Quantification steps (cycle 40 times) </br>a. 2 sec at x C to anneal primers. </br>Value of x depends on the average of the melting temperatures of the forward and reverse primers used. </br>b. 20 sec at 72 C to extend DNA. </br>c. 15 sec at 95 C to denature dsDNA. </br>3. Melting curve production </br>a. Start at 65 C to denature DNA. </br>b. Increase by 0.5 C/5 s until 65 C. </br>4. Single fluorescence reading during annealing step at each cycle during extension phase of the quantification step. </br>5. Fluorescence reading every 5 s during melting temperature determination.

</br></br><u>Data analysis outline for absolute quantification</u> </br>1. Create a standard curve. </br>a. Ensure that the efficiency is between 95% and 105%. </br>b. Ensure that the correlation coefficient is high. </br>2. Observe melting curve with standard samples to determine melting temperature for target DNA. </br>a. Single, sharp peaks denote single PCR product. </br>b. Multiple peaks in standard samples may mean primer dimers or interference from staples. </br>c. Estimate Tm of target theoretically to compare to experimental values.

<h3><u>6.3. WesternBlots</u></h3> </br><i>This protocol is optimized for ~1 x 107 cells.</i>

</br></br><strong>Reagents</strong> </br>● 1X Lysis buffer (100 mM Tris HCL pH 7.6; 0.5% Triton X-100, 1:100 200 mM PMSF/ in ethanol; 1:200 Protease inhibitor cocktail.) </br>● 1X Running buffer (25 mM Tris; 250 mM Glycine; 0.1% SDS;) </br>● 1X Transfer buffer (39 mM Glycine; 48 mM Tris-base; 0.037% SDS; 20% MeOH) </br>● 1L of 1X TBST (80 g NaCl; 30.25 Tris base; 2.24 g KCl; 500 uL Tween 20)

</br></br><strong>Protein quantification</strong> </br>● Wash cells twice in PBS. </br>● Remove supernatant and resuspend in 200-300 uL Lysis buffer. </br>● Incubate on ice for 20-30 minutes. </br>● Centrifuge at 12,000 rpm for 10 minutes. </br>● Use BCA protein microassay for protein concentrations. </br>○ Prepare BCA assay solution for all samples in a falcon tube </br>■ 200 uL reagent A for each sample, 1:50 dilution of reagent B. </br>○ Prepare standards by serially diluting bovine serum albumin to known concentrations. </br>○ In a 96 imaging plate, add 200 uL of BCA assay solution and 10 uL of sample or standard. </br>○ Incubate at 37 C for 30 min. </br>○ Image plate using a plate reader.

</br></br><strong>Polyacrylamide Gel Electrophoresis</strong> </br>● Prepare 4% SDS PAGE gel according to instructions. </br>● Place gel in apparatus and fill chamber with 1X Running buffer. </br>● Aliquot 4-12 ug of total protein extract and add 10 uL of Laemmli buffer solution to each sample. </br>● For biotinylated ladder, add 15 uL of Laemmli buffer solution to 1 uL of ladder. </br>● Heat samples at 100 C for 5 minutes. </br>● Load samples in well and run at 65 V until dye front enters resolving gel and then at 75 V for 2 - 3 hours.

</br></br><strong>Protein transfer to membrane</strong> </br>● Cut PVDF membrane to same size as gel. </br>● Soak membrane in methanol for 15 s, then H2O for 2 min, then transfer buffer for 5 min. </br>● Assemble transfer apparatus as below: </br>○ Red (+) current </br>○ Foam pad </br>○ Filter paper </br>○ PVDF </br>○ Gel </br>○ Filter paper </br>○ Foam pad </br>○ Black ( - ) current </br>● Upon assembly, flatten to remove any bubbles. </br>● Place assembly in apparatus, fill with transfer buffer, and run at 100 V for 1 hour, at 70 V for 3 hours, or at 14 V overnight.

</br></br><strong>Antibody blotting</strong> </br>● Block membrane with TBST + 5% dry milk with primary antibody (0.5-1 uL/mL) overnight at 4 C while rocking. </br>● Wash membrane thrice for 5 minutes with TBST rocking at room temperature. </br>● Blot membrane with secondary antibody (1:1000) in 10 mL TBST with 1% dry milk for 1 hour at room temperature while rocking. </br>● Repeat wash twice. </br>● Develop blot by semi-drying on paper towels, add Lumiglo (0.5 mL of each reagent into 9 mL water), incubate 20 s, and semi-dry on paper towels. </br>● Place face down onto plastic wrap in film cassette.

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Biomod/2014/OhioMOD/protocols: Difference between revisions

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@@ Line 387: / Line 387: @@
 </br>c.	Estimate Tm of target theoretically to compare to experimental values.
+<h3><u>6.3.	WesternBlots</u></h3>
+</br><i>This protocol is optimized for ~1 x 107 cells.</i>
+</br></br><strong>Reagents</strong>
+</br>●	1X Lysis buffer (100 mM Tris HCL pH 7.6; 0.5% Triton X-100, 1:100 200 mM PMSF/ in ethanol; 1:200 Protease inhibitor cocktail.)
+</br>●	1X Running buffer (25 mM Tris; 250 mM Glycine; 0.1% SDS;)
+</br>●	1X Transfer buffer (39 mM Glycine; 48 mM Tris-base; 0.037% SDS; 20% MeOH)
+</br>●	1L of 1X TBST (80 g NaCl; 30.25 Tris base; 2.24 g KCl; 500 uL Tween 20)
+</br></br><strong>Protein quantification</strong>
+</br>●	Wash cells twice in PBS.
+</br>●	Remove supernatant and resuspend in 200-300 uL Lysis buffer.
+</br>●	Incubate on ice for 20-30 minutes.
+</br>●	Centrifuge at 12,000 rpm for 10 minutes.
+</br>●	Use BCA protein microassay for protein concentrations.
+</br>○	Prepare BCA assay solution for all samples in a falcon tube
+</br>■	200 uL reagent A for each sample, 1:50 dilution of reagent B.
+</br>○	Prepare standards by serially diluting bovine serum albumin to known concentrations.
+</br>○	In a 96 imaging plate, add 200 uL of BCA assay solution and 10 uL of sample or standard.
+</br>○	Incubate at 37 C for 30 min.
+</br>○	Image plate using a plate reader.
+</br></br><strong>Polyacrylamide Gel Electrophoresis</strong>
+</br>●	Prepare 4% SDS PAGE gel according to instructions.
+</br>●	Place gel in apparatus and fill chamber with 1X Running buffer.
+</br>●	Aliquot 4-12 ug of total protein extract and add 10 uL of Laemmli buffer solution to each sample.
+</br>●	For biotinylated ladder, add 15 uL of Laemmli buffer solution to 1 uL of ladder.
+</br>●	Heat samples at 100 C for 5 minutes.
+</br>●	Load samples in well and run at 65 V until dye front enters resolving gel and then at 75 V for 2 - 3 hours.
+</br></br><strong>Protein transfer to membrane</strong>
+</br>●	Cut PVDF membrane to same size as gel.
+</br>●	Soak membrane in methanol for 15 s, then H2O for 2 min, then transfer buffer for 5 min.
+</br>●	Assemble transfer apparatus as below:
+</br>○	Red (+) current
+</br>○	Foam pad
+</br>○	Filter paper
+</br>○	PVDF
+</br>○	Gel
+</br>○	Filter paper
+</br>○	Foam pad
+</br>○	Black ( - ) current
+</br>●	Upon assembly, flatten to remove any bubbles.
+</br>●	Place assembly in apparatus, fill with transfer buffer, and run at 100 V for 1 hour, at 70 V for 3 hours, or at 14 V overnight.
+</br></br><strong>Antibody blotting</strong>
+</br>●	Block membrane with TBST + 5% dry milk with primary antibody (0.5-1 uL/mL) overnight at 4 C while rocking.
+</br>●	Wash membrane thrice for 5 minutes with TBST rocking at room temperature.
+</br>●	Blot membrane with secondary antibody (1:1000) in 10 mL TBST with 1% dry milk for 1 hour at room temperature while rocking.
+</br>●	Repeat wash twice.
+</br>●	Develop blot by semi-drying on paper towels, add Lumiglo (0.5 mL of each reagent into 9 mL water), incubate 20 s, and semi-dry on paper towels.
+</br>●	Place face down onto plastic wrap in film cassette.