mirror of
https://github.com/scinote-eln/scinote-web.git
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1088 lines
50 KiB
JSON
1088 lines
50 KiB
JSON
{
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"experiment": {
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"id": 497,
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"name": "SDS-PAGE for Protein Characterization",
|
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"description": "This experiment template will help you separate proteins based on their molecular weight. \r\nProteins are separated by electrophoresis through a gel matrix. Smaller proteins migrate faster due to less resistance from the gel matrix. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins. In SDS-PAGE, the use of sodium dodecyl sulfate (SDS, also known as sodium lauryl sulfate) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length.\r\nStaining with Coomassie blue dyes is commonly used to stain proteins and visualize them. But if you want to identify specific proteins (using specific antibodies) a better technique to use is a Western blot.",
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"id": 2388,
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"name": "Western Blot",
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{
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"step": {
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"id": 5264,
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"name": "Probing with antibodies and detection of the bands",
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||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p><strong>Blocking buffer: </strong></p>\n<ul>\n<li>\n<strong>1-3% BSA in PBS-T: </strong>Bovine serum albumin (BSA) is used as a blocking agent.</li>\n<li>\n<strong>1-5% skim milk in PBS-T </strong>Skim milk is more effective in blocking and used frequently. However, it can interfere with specific antigen-antibody reactions. <em><strong>Skim milk contains the </strong><strong>phosphoprotein </strong><strong>casein and therefore is not suitable for the detection of phosphoproteins.</strong></em>\n</li>\n</ul>\n</body></html>",
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"checklist_items": [
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{
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"id": 4013,
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"text": "Place the membrane in blocking buffer and incubate at room temperature for 1 hour (or at 4°C overnight) with shaking.",
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"checked": false,
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"checklist_id": 974,
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"created_at": "2018-12-24T13:33:23.310Z",
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},
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{
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"id": 4014,
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"text": "Wash the membrane with washing buffer 3 times (5 minutes each time).",
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||
"checked": false,
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||
"checklist_id": 974,
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"created_at": "2018-12-24T13:33:23.316Z",
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"position": 1
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},
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{
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"id": 4015,
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"text": "Place the membrane in primary antibody solution diluted in 1% BSA in Tween PBS, pH 7.2, and incubate at room temperature for 1 hour with shaking.",
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"checked": false,
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"checklist_id": 974,
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"created_at": "2018-12-24T13:33:23.321Z",
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"position": 2
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},
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{
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"id": 4016,
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"text": "Wash the membrane with washing buffer 3 times (10 minutes each time).",
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"checked": false,
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"checklist_id": 974,
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"created_at": "2018-12-24T13:33:23.327Z",
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"position": 3
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},
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{
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"id": 4017,
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"text": "Place the membrane in secondary antibody diluted in 1% BSA in Tween PBS, pH 7.2, and incubate at room temperature for 1 hour with shaking.",
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||
"checked": false,
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||
"checklist_id": 974,
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"created_at": "2018-12-24T13:33:23.332Z",
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"updated_at": "2018-12-24T13:33:23.332Z",
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"position": 4
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},
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{
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"id": 4018,
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"text": "Wash the membrane with washing buffer 3 times (10 minutes each time).",
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||
"checked": false,
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"checklist_id": 974,
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"created_at": "2018-12-24T13:33:23.337Z",
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"position": 5
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}
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]
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}
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],
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{
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"step": {
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"id": 5262,
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"name": "Transfer of proteins to a membrane",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Soak a piece of <strong>PVDF membrane</strong> slightly larger than the gel in methanol for <strong>1 minute</strong> (pre-wetting), and then equilibrate in transfer buffer. Also, equilibrate two pieces of filter paper slightly larger than the PVDF membrane in transfer buffer. There is no need for pre-wetting a nitrocellulose membrane. Skip using methanol, and equilibrate the membrane in transfer buffer.<br><br><strong>Transfer buffer:</strong></p>\n<ul>\n<li>25 mM Tris-HCl</li>\n<li>192 mM glycine</li>\n<li>20% MeOH</li>\n</ul>\n<br>Equilibrate the gel in transfer buffer after electrophoresis. <br><br>Place a piece of equilibrated filter paper on the anode plate, and place the membrane, gel, and another piece of filter paper without any air bubbles. All of these should be sandwiched between two sponges.</body></html>",
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"created_at": "2018-12-24T13:17:10.122Z",
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"updated_at": "2019-02-20T07:30:20.884Z",
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"created_at": "2019-02-20T07:29:55.862Z",
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{
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"step": {
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"id": 5263,
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"name": "Blotting",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Connect <strong>the plus electrode to the anode plate</strong> and connect the <strong>negative electrode to the cathode plate</strong>.<br>Turn on the power supply and transfer for <strong>1 hour</strong> at <strong>50 mA</strong> (for one gel).<br><br>Turn off the power supply, remove the membrane, and temporarily stain to check the efficiency of transfer and to visualize the molecular weight markers to confirm their positions.<br><strong>Ponceau S</strong> is commonly used for this purpose because the membrane is easily destained, and it does not interfere with subsequent probing with antibodies.<br><br><strong>Ponceau S solution: </strong></p>\n<ul>\n<li>1g Ponceau S</li>\n<li>50mL acetic acid</li>\n<li>Make up to 1L with ddH2O</li>\n</ul>\n</body></html>",
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"position": 1,
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"created_at": "2018-12-24T13:24:09.108Z",
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},
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{
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"step": {
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||
"id": 5265,
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"name": "Chemiluminescence method",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Prepare the chemiluminescence detection reagent according to the instruction manual. Place the membrane on a piece of plastic wrap, and add the reagent to the membrane. Allow it to stand for <strong>1 minute</strong>, and remove the reagent using a pipette. <br>Wrap the membrane in a piece of plastic wrap, remove any air bubbles, and place it under a chemiluminescence detection apparatus (e.g., CCD camera) for viewing.</p></body></html>",
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"created_at": "2018-12-24T13:34:41.460Z",
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},
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{
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"id": 2387,
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"name": "Standard Coomassie Stain",
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"steps": [
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"step": {
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"id": 5260,
|
||
"name": "Destain",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<strong>Destain solution</strong>: Add <strong>500mL</strong> of HPLC- grade methanol to <strong>300 mL</strong> of HPLC grade water. Add <strong>100 mL</strong> of reagent grade acetic acid and, after mixing, adjust the total volume to <strong>1000mL</strong> with water. The final concentrations are <strong>50%</strong> (v/v) methanol in water with <strong>10%</strong> (v/v) acetic acid. <br><br>Cover the gel with<strong> ~250mL</strong> of the destain solution and allow the gel to destain with gentle agitation. The destain solution should be changed several times, removing it at each change by aspiration. Continue the destaining until the protein bands are seen without background staining of the gel.</body></html>",
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"step": {
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||
"id": 5258,
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||
"name": "Washing of the gel",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Cover the gel with <strong>500mL</strong> of the gel-washing solution. <br><br><strong>Gel-washing solution:</strong> Add <strong>500mL</strong> of HPLC-grade methanol to <strong>300 mL</strong> of HPLC grade water. Add <strong>100mL</strong> of reagent grade acetic acid and adjust the total volume to <strong>1000 mL</strong> with HPLC grade water. The final concentrations are <strong>50%</strong> (v/v) methanol in water with <strong>10%</strong> (v/v) acetic acid. <br><br>Continue to fix the proteins in the gel by incubating overnight at room temperature with gentle agitation. The gel should be covered during this process to avoid contamination and to prevent the evaporation of the solution. At the end of this time, remove the solution by aspiration.</p></body></html>",
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"created_at": "2018-12-24T12:47:54.498Z",
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"updated_at": "2019-01-29T10:09:33.373Z",
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"step": {
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||
"id": 5259,
|
||
"name": "Comassie staining",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p><strong>Comassie Stain:</strong> Dissolve <strong>0.4g</strong> of Coomassie blue R350 in <strong>200 mL</strong> of <strong>40%</strong> (v/v) HPLC grade methanol in water with stirring as needed. Filter the solution to remove any insoluble material. Add <strong>200mL</strong> of <strong>20%</strong> (v/v) acetic acid in water. The final concentration is <strong>0.1%</strong> (w/v) Coomassie blue R350, <strong>20%</strong> (v/v) methanol, and <strong>10%</strong> (v/v) acetic acid.<br><br>Cover the gel with <strong>400mL</strong> of the Coomassie stain. Stain the gel at room temperature for <strong>3 to 4 hours</strong> with gentle agitation. The Coomassie stain is removed by aspiration after staining.</p></body></html>",
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"created_at": "2018-12-24T12:49:39.311Z",
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"updated_at": "2019-01-29T10:19:56.594Z",
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||
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|
||
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|
||
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||
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||
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|
||
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|
||
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|
||
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|
||
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||
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||
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||
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|
||
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||
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||
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||
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||
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||
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||
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||
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||
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|
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|
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|
||
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|
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|
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||
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||
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||
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|
||
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|
||
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|
||
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|
||
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|
||
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|
||
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||
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|
||
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|
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||
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||
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|
||
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|
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|
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|
||
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||
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||
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||
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||
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||
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|
||
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|
||
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|
||
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||
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||
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"step": {
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"name": "Make the stacking gel",
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"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Proteins are highly concentrated when they migrate through a stacking gel prior to entering a separating gel. The concentration occurs due to the difference in the rate of migration of glycine ion, chloride ion, and proteins.<strong><br><br>5mL stacking gel:</strong></p>\n<ul>\n<li>H2O 2.975 mL</li>\n<li>0.5 M Tris-HCl, pH 6.8 1.25 mL</li>\n<li>10% (w/v) SDS 0.05 mL</li>\n<li>Acrylamide/Bis-acrylamide (30%/0.8% w/v) 0.67 mL</li>\n<li>10% (w/v) ammonium persulfate (AP) 0.05 mL</li>\n<li>TEMED 0.005 mL</li>\n</ul>\n<strong><strong>1x Running buffer:<br></strong></strong>\n<ul>\n<li>25 mM Tris-HCl</li>\n<li>200 mM Glycine</li>\n<li>0.1% (w/v) SDS</li>\n</ul>\n</body></html>",
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"position": 2,
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"checklists": [
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{
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"checklist": {
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"id": 973,
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"name": "Guideline",
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},
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"checklist_items": [
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{
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"id": 4007,
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"text": "Discard the water and you can see separating gel left.",
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"checked": false,
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"created_at": "2018-12-24T11:30:55.054Z",
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},
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{
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"id": 4008,
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"text": "Pipette in stacking gel until an overflow.",
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"created_at": "2018-12-24T11:30:55.060Z",
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},
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{
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"id": 4009,
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||
"text": "Insert the well-forming comb without trapping air under the teeth. Wait for 20-30min to let it gelate.",
|
||
"checked": false,
|
||
"checklist_id": 973,
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"created_at": "2018-12-24T11:30:55.065Z",
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},
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{
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"id": 4010,
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"text": "Make sure a complete gelation of the stacking gel and take out the comb.",
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"checked": false,
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"created_at": "2018-12-24T11:30:55.071Z",
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"position": 3
|
||
},
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||
{
|
||
"id": 4011,
|
||
"text": "Take the glass plates out of the casting frame and set them in the cell buffer dam.",
|
||
"checked": false,
|
||
"checklist_id": 973,
|
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"created_at": "2018-12-24T11:30:55.076Z",
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"position": 4
|
||
},
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||
{
|
||
"id": 4012,
|
||
"text": "Pour the running buffer (electrophoresis buffer) into the inner chamber and keep pouring after overflow untill the buffer surface reaches the required level in the outer chamber.",
|
||
"checked": false,
|
||
"checklist_id": 973,
|
||
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|
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||
],
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"asset": {
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|
||
"lock": null,
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|
||
},
|
||
"asset_blob": {
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"filename": "Gel_Preparation.jpg"
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}
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}
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],
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"step_tables": [],
|
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|
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|
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}
|
||
],
|
||
"results": []
|
||
},
|
||
{
|
||
"my_module": {
|
||
"id": 2384,
|
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"name": "Sample preparation",
|
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|
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|
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|
||
"state": "uncompleted",
|
||
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|
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},
|
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"outputs": [
|
||
{
|
||
"id": 6047,
|
||
"input_id": 2386,
|
||
"output_id": 2384
|
||
}
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"nr_of_linked_children": 0
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},
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"protocol_protocol_keywords": [],
|
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"steps": [
|
||
{
|
||
"step": {
|
||
"id": 5253,
|
||
"name": "Centrifugation",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<strong>Centrifuge:</strong><br><br>\n<ul>\n<li>15,000 rpm</li>\n<li>1 minute</li>\n<li>4°C</li>\n</ul>\n<br>Use <strong>the supernatant</strong> for SDS-PAGE.</body></html>",
|
||
"position": 2,
|
||
"completed": false,
|
||
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|
||
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||
"created_at": "2018-12-24T11:48:19.756Z",
|
||
"updated_at": "2019-02-19T14:03:57.459Z",
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|
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},
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||
{
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||
"step": {
|
||
"id": 5252,
|
||
"name": "Heat the samples",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Heat the samples at <strong>100°C</strong> for <strong>3 minutes</strong> in a heat block.</p></body></html>",
|
||
"position": 1,
|
||
"completed": false,
|
||
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|
||
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"created_at": "2018-12-24T11:47:05.158Z",
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||
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},
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"tables": []
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},
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||
{
|
||
"step": {
|
||
"id": 5251,
|
||
"name": "Mix samples with buffer",
|
||
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<p>Make sure your target protein is <strong>dissolved in the liquid phase</strong>, and <strong>no inappropriate ingredients are present</strong> (e.g. guanidine hydrochloride can interact with SDS and cause precipitation). Generally, to treat your unprepared sample, you can use sonicator, lysis buffer or both to sufficiently make your target protein released, and centrifuge to make supernatant and pellet separated.<br><br><br>Mix samples with loading buffer. Mix by flicking the tube.<strong><br><br>5X Sample buffer (Loading buffer):</strong></p>\n<ul>\n<li>10% w/v SDS</li>\n<li>10 mM Dithiothreitol, or beta-mercapto-ethanol</li>\n<li>20 % v/v Glycerol</li>\n<li>0.2 M Tris-HCl; pH 6.8</li>\n<li>0.05% w/v Bromophenolblue</li>\n</ul>\n<strong><br><br><br><br><br><br><br></strong>\n</body></html>",
|
||
"position": 0,
|
||
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|
||
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||
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||
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}
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}
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],
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}
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],
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}
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}
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