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scinote-web/app/assets/templates/experiment_454/experiment.json
Urban Rotnik 41cd8edf29 Fix 'bellow' typo across scinote
2021-02-17 11:56:30 +01:00

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{
"experiment": {
"id": 454,
"name": "Bottom-up Proteomics",
"description": "This template will help you prepare protein samples, followed by the cleanup and mass spectrometry analysis. \r\nIn bottom-up proteomics, the protein is first broken up into peptides, either by chemical or enzymatic digestion. In the first step, sample preparation is required after gel electrophoresis, liquid chromatography or affinity capture. Samples can (optionally) be enriched at the peptide level. With mass spectrometry, in the end, you can identify and characterize biological molecules.",
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"updated_at": "2019-01-29T09:46:44.060Z",
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},
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{
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},
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},
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{
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"name": "Select appropriate MS Instrument",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Depending on the different type of samples there are different mass spectrometry instrument that can be selected. </p></body></html>",
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},
{
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"name": "MS Data Analysis",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Differences among search engines can include the scoring algorithm, whether it considers data quality, and whether it considers all fragment ion types or only a subset. Often, the best way to select an algorithm is to examine the details regarding data-format compatibility and necessary features, test it on a data set, and compare the results to what is expected. <br><br></p></body></html>",
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},
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}
],
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},
"asset_blob": {
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}
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},
{
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"restored_by_id": null,
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},
"outputs": [
{
"id": 5978,
"input_id": 2345,
"output_id": 2258
}
],
"my_module_tags": [],
"task_comments": [],
"my_module_repository_rows": [],
"user_my_modules": [],
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{
"step": {
"id": 5146,
"name": "Perform enzymatic digestion",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div style=\"text-align: justify;\">The protein is cut enzymatically into a limited number of shorter fragments during digestion and these fragments are called peptides and allow for the identification of the protein with their characteristic mass and pattern.</div></body></html>",
"position": 5,
"completed": false,
"completed_on": null,
"user_id": 202,
"created_at": "2018-12-07T11:40:40.624Z",
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"checklist_items": [
{
"id": 3869,
"text": "Dilute the gel enzyme stock solution ~1:1000 with 25 mM ammonium bicarbonate to obtain a 10 to 20 μg/mL working solution.",
"checked": false,
"checklist_id": 942,
"created_at": "2018-12-20T14:15:25.671Z",
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"position": 0
},
{
"id": 3870,
"text": "Add a sufficient amount of the gel enzyme working solution to cover gel pieces and incubate on ice for 1 hr. For a typical gel band/spot, this is ~10 to 20 μL.",
"checked": false,
"checklist_id": 942,
"created_at": "2018-12-20T14:15:25.679Z",
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"position": 1
},
{
"id": 3871,
"text": "Remove excess gel enzyme solution, then add sufficient 25 mM NH4HCO3 to cover the gel pieces. This increases the pH and thereby inhibits the enzymatic digestion for those enzymes which have low pH optima.",
"checked": false,
"checklist_id": 942,
"created_at": "2018-12-20T14:15:25.686Z",
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},
{
"id": 3872,
"text": "Incubate at 37°C overnight.",
"checked": false,
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"created_at": "2018-12-20T14:15:25.693Z",
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"step": {
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"name": "Reduce disulfide bonds and alkylate free cysteines",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<em><strong>Dithiothreitol (DTT) is a reducing agent that converts cystine’s disulfide bond into cysteine’s free sulfhydryl groups.</strong></em><br><em><strong>Iodoacetamide (IAA) is an alkylating agent that reacts with free sulfhydryl groups of cysteine residues to form S-</strong></em><strong><em>carboxyamidomethyl-cysteine, which cannot be reoxidized to form disulfide bonds. This is important for allowing trypsin maximum access to cleavage sites within the protein.</em></strong>\n</body></html>",
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{
"id": 3865,
"text": "Add 100 μL of 10 mM dithiothreitol to the gel piece, then incubate 45 min at 55°C.",
"checked": false,
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"created_at": "2018-12-20T14:12:18.062Z",
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{
"id": 3866,
"text": "Remove solution, add 100 μl 55 mM iodoacetamide, and incubate 30 min at room temperature in the dark.",
"checked": false,
"checklist_id": 941,
"created_at": "2018-12-20T14:12:18.070Z",
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{
"id": 3867,
"text": "Remove IAA solution and add 400 μL gel wash solution, incubate at room temperature, and shake for 15 min. Repeat this step twice.",
"checked": false,
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"created_at": "2018-12-20T14:12:18.077Z",
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"position": 2
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{
"id": 3868,
"text": "Dehydrate gel pieces with 400 μL of 100% acetonitrile for 10 min, then remove supernatant and dry gel pieces using a vacuum centrifuge.",
"checked": false,
"checklist_id": 941,
"created_at": "2018-12-20T14:12:18.083Z",
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"step": {
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"name": "Extract peptides",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Here is the guideline how to extract peptides. This step can take up to several hours depending upon the volume used.</p></body></html>",
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{
"id": 3873,
"text": "Remove supernatant, which now contains peptides, to a new clean microcentrifuge tube.",
"checked": false,
"checklist_id": 943,
"created_at": "2018-12-20T14:17:36.754Z",
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{
"id": 3874,
"text": "Add 100 μL gel extraction solution to the gel pieces and incubate while shaking for 20 min at room temperature.",
"checked": false,
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{
"id": 3875,
"text": "Remove solution and combine with the solution obtained in step 1.",
"checked": false,
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"text": "Repeat steps second and third step.",
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"position": 3
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{
"id": 3877,
"text": "Dry combined supernatants using a vacuum centrifuge.",
"checked": false,
"checklist_id": 943,
"created_at": "2018-12-20T14:17:36.791Z",
"updated_at": "2018-12-20T14:17:36.791Z",
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"step": {
"id": 4826,
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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<em><strong>Coomassie dyes (also known as Coomassie Brilliant Dyes) are a family of dyes commonly used to stain proteins in sodium dodecyl sulfate and blue native polyacrylamide gel electrophoresis (SDS-PAGE and BN-PAGE, respectively) gels.</strong></em> The gels are soaked in dye and an excess stain is then eluted with a solvent. This treatment allows the visualization of protein bands.</body></html>",
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{
"id": 3780,
"text": "Remove destain solution",
"checked": false,
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"position": 0
},
{
"id": 3781,
"text": "Add 400 μl water.",
"checked": false,
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"updated_at": "2018-12-07T10:52:29.554Z",
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"position": 1
},
{
"id": 3782,
"text": "If the gel pieces are still blue, remove the water and repeat until the gel pieces are colourless.",
"checked": false,
"checklist_id": 917,
"created_at": "2018-12-07T10:52:29.562Z",
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},
"checklist_items": [
{
"id": 3786,
"text": "Dehydrate gel pieces with 400 μl of 100% acetonitrile for 10 min",
"checked": false,
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"created_at": "2018-12-07T11:39:18.118Z",
"updated_at": "2018-12-07T11:39:18.118Z",
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"position": 0
},
{
"id": 3787,
"text": "Remove supernatant",
"checked": false,
"checklist_id": 919,
"created_at": "2018-12-07T11:39:18.126Z",
"updated_at": "2018-12-07T11:39:18.126Z",
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"position": 1
},
{
"id": 3788,
"text": "Dry gel pieces in a vacuum centrifuge",
"checked": false,
"checklist_id": 919,
"created_at": "2018-12-07T11:39:18.133Z",
"updated_at": "2018-12-07T11:39:18.133Z",
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}
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"step": {
"id": 4824,
"name": "Destain gel bands",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p>Destain in the microcentrifuge tube for <strong>30 min</strong> with <strong>100 μL</strong> of the appropriate gel destain solution with vigorous vortexing. The choice of destaining protocol depends on the stain used, and there is a specific destain recipe for each type of gel stain. The choice of protein stain depends on the protein concentration and the dynamic range of the sample. Silver and colloidal Coomassie staining (CCS) stains offer a low limit of detection (<strong>1 to 10 ng</strong>) compared to traditional Coomassie staining (<strong>0.3 to 1 μg)</strong>.</p></body></html>",
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"created_at": "2018-11-26T13:08:09.568Z",
"updated_at": "2019-01-31T08:32:58.429Z",
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"checklist": {
"id": 940,
"name": "Follow:",
"step_id": 4824,
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},
"checklist_items": [
{
"id": 3863,
"text": "Step 3: Glutaraldehyde-free silver stained gels",
"checked": false,
"checklist_id": 940,
"created_at": "2018-12-20T14:03:52.362Z",
"updated_at": "2018-12-20T14:04:56.749Z",
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"position": 0
},
{
"id": 3864,
"text": "Step 4: Coomassie-stained gels",
"checked": false,
"checklist_id": 940,
"created_at": "2018-12-20T14:03:52.369Z",
"updated_at": "2018-12-20T14:04:56.761Z",
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"step": {
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"name": "Silver stained gels",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body>\n<em><strong>Silver staining is used to detect proteins after electrophoretic separation on polyacrylamide gels.</strong></em> It combines excellent sensitivity, whilst using very simple and cheap equipment and chemicals.</body></html>",
"position": 2,
"completed": false,
"completed_on": null,
"user_id": 202,
"created_at": "2018-11-26T13:14:10.347Z",
"updated_at": "2018-12-24T06:30:40.360Z",
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},
"checklist_items": [
{
"id": 3776,
"text": "Remove destain solution",
"checked": false,
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"created_at": "2018-12-07T10:50:00.148Z",
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"position": 0
},
{
"id": 3777,
"text": "Wash gel piece with 400 μL water",
"checked": false,
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"created_at": "2018-12-07T10:50:00.157Z",
"updated_at": "2018-12-20T12:00:27.604Z",
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"position": 1
},
{
"id": 3778,
"text": "Shake for 15 min at room temperature",
"checked": false,
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"created_at": "2018-12-07T10:50:00.166Z",
"updated_at": "2018-12-07T10:50:00.166Z",
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"position": 2
},
{
"id": 3779,
"text": "Repeat wash at least twice or until gel pieces are colourless.",
"checked": false,
"checklist_id": 916,
"created_at": "2018-12-07T10:50:00.175Z",
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"checklist_items": [
{
"id": 3783,
"text": "Dehydrate gel pieces with 400 μL of 100% acetonitrile for 10 min",
"checked": false,
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"updated_at": "2018-12-20T12:00:27.594Z",
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"position": 0
},
{
"id": 3784,
"text": "Remove supernatant",
"checked": false,
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"created_at": "2018-12-07T11:36:41.204Z",
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"position": 1
},
{
"id": 3785,
"text": "Dry gel pieces in a vacuum centrifuge",
"checked": false,
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"created_at": "2018-12-07T11:36:41.211Z",
"updated_at": "2018-12-07T11:36:41.211Z",
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"position": 2
}
]
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{
"step": {
"id": 4823,
"name": "Excise gel bands",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><p style=\"text-align: justify;\">Once you have run your gel, move it to an<strong> open UV box</strong> (be sure to wear proper UV protection - especially for your eyes!), remove it from any gel tray as plastic will block much of the UV and with a clean, sterile razor blade, slice the desired DNA fragment from the gel. Try to get as little excess gel around the band as possible. To do so, it is often important to take the excised band, lay it down on the UV box and trim the top, bottom and sides with the razor blade. This is especially important during the DNA purification step, as many kits cannot handle more than a certain total volume of gel per reaction.<br><br></p></body></html>",
"position": 0,
"completed": false,
"completed_on": null,
"user_id": 202,
"created_at": "2018-11-26T13:01:32.320Z",
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"created_at": "2019-02-20T07:32:07.412Z",
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"last_modified_by_id": null,
"estimated_size": 12461,
"lock": null,
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"version": 1,
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"asset_blob": {
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},
{
"my_module": {
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"due_date": null,
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"x": 0,
"y": 16,
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"workflow_order": 1,
"experiment_id": 454,
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}
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"steps": [
{
"step": {
"id": 4840,
"name": "Neutralization of samples",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div style=\"text-align: justify;\">The pH of the sample may be neutralized either prior to or after drying via vacuum centrifugation to remove acetonitrile and trifluoroacetic acid. <span style=\"text-align: left;\">RP-HPLC samples will likely contain aqueous trifluoroacetic acid and acetonitrile since these are the most commonly used mobile phases. </span><span style=\"text-align: left;\">The NH</span><sub style=\"text-align: left;\">4</sub><span style=\"text-align: left;\">HCO</span><sub style=\"text-align: left;\">3</sub><span style=\"text-align: left;\"> solution should be prepared fresh, as the pH of the solution will increase with age at room temperature. It is possible to store for several days at <strong>4°C</strong>, but the pH should be checked prior to use.</span>\n</div></body></html>",
"position": 0,
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"checklist": {
"id": 944,
"name": "To neutralize prior to drying:",
"step_id": 4840,
"created_at": "2018-12-20T14:20:33.276Z",
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"checklist_items": [
{
"id": 3878,
"text": "Add sufficient 1 M NH4HCO3, pH8.5.",
"checked": false,
"checklist_id": 944,
"created_at": "2018-12-20T14:20:33.278Z",
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"position": 0
},
{
"id": 3879,
"text": "Adjust final pH to ~8.0.",
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},
{
"id": 3880,
"text": "Then dry via vacuum centrifugation.",
"checked": false,
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{
"id": 3881,
"text": "Resuspend dried protein in 100 mM NH4HCO3, pH 8.5.",
"checked": false,
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},
{
"id": 3882,
"text": "Spot an aliquot of the neutralized solution on narrow-range pH paper to validate that neutralization (to pH 8.0) has occurred.",
"checked": false,
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"step": {
"id": 5148,
"name": "Reduction of disulfide bonds and alkyle free cysteine residues",
"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><em><strong>Reduction of disulfide bonds can be achieved with either dithiothreitol (DTT) or tris (2-carboxyethyl)phosphine hydrochloride (TCEP). </strong></em>DTT works optimally at <strong>pH 7 to 9</strong>, TCEP is typically more efficient, works at a wider pH range (<strong>pH 2 to 11</strong>), and has no offensive odour. The concentrations and incubation times for DTT, TCEP, and IAA may vary depending on the amount of protein to be treated. Typical concentrations range from <strong>5 to 10 mM</strong> for DTT and TCEP, and <strong>10 to 50 mM</strong> for iodoacetamide. Incubation times may range from <strong>5 min to 1 hr for the reduction step</strong> and <strong>10 min to 1 hr for the alkylation step</strong>. Similarly, the reduction can be performed at room temperature, <strong>37°C or 56°C</strong>. Each parameter can be optimized depending on the nature of the protein sample.</p>\n<div><em>Optional: Following alkylation, adjust the sample to <strong>40 mM</strong> DTT.</em></div>\n</body></html>",
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"completed": false,
"completed_on": null,
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"created_at": "2018-12-07T12:46:55.270Z",
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"created_at": "2018-12-20T14:24:01.663Z",
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"checklist_items": [
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"id": 3883,
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"checked": false,
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"created_at": "2018-12-20T14:24:01.680Z",
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"position": 1
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{
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"text": "Add iodoacetamide (IAA) to a concentration of 10 mM final and place at room temperature in the dark for 30 min.",
"checked": false,
"checklist_id": 946,
"created_at": "2018-12-20T14:24:01.690Z",
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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<div style=\"text-align: justify;\">Digest the protein sample into peptides using an <strong>appropriate enzyme</strong> (e.g., chymotrypsin, trypsin, LysC, or AspN) or<strong> chemical</strong> (CNBr/70% formic acid). If using trypsin, add sufficient enzyme for final trypsin: protein ratio of <strong>1:20 to 1:100</strong> (w/w). Vortex briefly, seal the tube with Parafilm and incubate with end-over-end rotation at <strong>37°C</strong> for <strong>4 to 18 hours</strong>.</div>\n<div style=\"text-align: justify;\">\n<br>The amount of trypsin needed will vary depending on the amount of protein sample present and the desired speed of digestion. To minimize trypsin autolysis, which will add extra peaks to the MS spectra, use the minimum amount of trypsin.<strong> Typically, a ratio of 1:20 (w/w) is sufficient for complete digestion of 10 μg protein within 4 hr</strong>. If protein concentration is especially high or the accessibility of trypsin to the cleavage sites is hampered (e.g., due to insolubility), it may be helpful to add another aliquot of trypsin or perform short digestion (<strong>4 hours</strong>) with LysC prior to the addition of trypsin. Finally, the activity of trypsin is enhanced in the presence of acetonitrile (<strong>10% to 50% v/v</strong>), and, thus, acetonitrile may be added to aid in the solubilization of the protein during tryptic digestion.</div>\n<div style=\"text-align: justify;\">\n<br><strong>Trypsin activity is greatest at pH 8.0</strong>. Thus, it is recommended that the pH of the solution be checked prior to the addition of the enzyme. In cases where sample volume is small, a <strong>1- to 2-μL </strong>sample may be tested using pH paper. Stop reaction by adding <strong>5 μL</strong> of<strong> 1.0%</strong> trifluoroacetic (TFA) acid (pH after TFA addition should be <strong>~2 to 3</strong>).</div>\n</body></html>",
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"created_at": "2018-12-07T12:52:36.364Z",
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"step": {
"id": 5154,
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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><p>Follow elution and collection guidelines below.</p></body></html>",
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"checklist": {
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"name": "Guidelines:",
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"created_at": "2018-12-20T14:51:18.465Z",
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"created_by_id": null,
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"checklist_items": [
{
"id": 3901,
"text": "If samples have been dried, then resuspended in 3 μL 1 of % SDS. Dilute IMAC–1% TFA and IMAC-FT samples each in 5 volumes of TiO2 loading solution.",
"checked": false,
"checklist_id": 951,
"created_at": "2018-12-20T14:51:18.467Z",
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"position": 0
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"id": 3902,
"text": "Load each of the samples onto one TiO2 column (for simple samples) or three to four TiO2 columns (for complex mixtures containing ≥120 μg protein).",
"checked": false,
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"created_at": "2018-12-20T14:51:18.476Z",
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"position": 1
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"id": 3903,
"text": "Collect TiO2 flow through in microcentrifuge tubes. Do this step slowly using Combi-Syringe to apply the pressure.",
"checked": false,
"checklist_id": 951,
"created_at": "2018-12-20T14:51:18.483Z",
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"position": 2
},
{
"id": 3904,
"text": "Wash the TiO2 columns using 5 μL TiO2 loading solution and pool the eluates with the TIO2FT fraction from the previous step. Do this step slowly using Combi-Syringe to apply the pressure.",
"checked": false,
"checklist_id": 951,
"created_at": "2018-12-20T14:51:18.491Z",
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"created_by_id": null,
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"position": 3
},
{
"id": 3905,
"text": "Elute the phosphopeptides using 30 μL high-pH elution solution. Use 30 μL per column and pool the eluates from the same sample. This step should be performed slowly using a Combi-Syringe to apply pressure.\r\nAcidify the eluates using 100% TFA to a pH of ~2 to 3.",
"checked": false,
"checklist_id": 951,
"created_at": "2018-12-20T14:51:18.499Z",
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"step": {
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"checklists": [
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"checklist": {
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"name": "For MALDI-TOF-MS/MS:",
"step_id": 5156,
"created_at": "2018-12-07T14:34:54.228Z",
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"checklist_items": [
{
"id": 3789,
"text": "Elute the peptides from Oligo R3 column using 5 μL phosphopeptide RP elution buffer",
"checked": false,
"checklist_id": 920,
"created_at": "2018-12-07T14:34:54.230Z",
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"position": 0
},
{
"id": 3790,
"text": "Spot directly onto MALDI sample plate, using a pipet.",
"checked": false,
"checklist_id": 920,
"created_at": "2018-12-07T14:34:54.239Z",
"updated_at": "2018-12-07T14:34:54.239Z",
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"position": 1
},
{
"id": 3791,
"text": "As spot is drying, if matrix crystals are not forming well, spot an additional 0.5 μL phosphopeptide RP elution buffer.",
"checked": false,
"checklist_id": 920,
"created_at": "2018-12-07T14:34:54.248Z",
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"position": 2
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"checklist": {
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"name": "For LC-ESI-MS/MS:",
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"created_at": "2018-12-07T14:34:54.261Z",
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"checklist_items": [
{
"id": 3792,
"text": "Elute the peptides from the Oligo R3 column using 30 μl phosphopeptide RP elution buffer.",
"checked": false,
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"created_at": "2018-12-07T14:34:54.263Z",
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"position": 0
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"id": 3793,
"text": "Dry pooled eluates by vacuum centrifugation.",
"checked": false,
"checklist_id": 921,
"created_at": "2018-12-07T14:34:54.271Z",
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},
{
"id": 3794,
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"checked": false,
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"created_at": "2018-12-07T14:34:54.278Z",
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"step": {
"id": 5155,
"name": "Preparation of a slurry of Oligo R3 reversed-phase resin",
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{
"step": {
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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<div style=\"text-align: justify;\">Prepare a TiO<sub>2</sub> bead slurry in <strong>100%</strong> acetonitrile. Pack the TiO<sub>2</sub> in StageTips that have been pre-packed with C<sub>8</sub> disks. The resulting TiO<sub>2</sub> columns should be <strong>~4 to 5 mm</strong> long.</div>\n<div style=\"text-align: justify;\"><em><strong>TiO<sub>2</sub> is light sensitive, so keep the powder in an amber or dark glass container or in a microcentrifuge tube covered with aluminium foil. When not in use, TiO<sub>2</sub> should be in a container stored in a dark place (e.g., a drawer). It should not be exposed to light for longer than necessary.</strong></em></div>\n</body></html>",
"position": 0,
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"step": {
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"checklist_items": [
{
"id": 3897,
"text": "Collect the column flow through in microcentrifuge tubes.",
"checked": false,
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"position": 0
},
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"id": 3898,
"text": "Wash the IMAC column with 50 μL IMAC wash solution and pool the eluate with the fraction from the previous step. This need to be done slowly using a Combi-Syringe to apply pressure.",
"checked": false,
"checklist_id": 950,
"created_at": "2018-12-20T14:48:32.256Z",
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"text": "Elute and collect the monophosphopeptides using 50 μl low-pH elution solution. This needs to be done quickly using a Combi-Syringe to apply pressure.",
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"id": 3900,
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"created_at": "2018-12-20T14:48:32.269Z",
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"id": 3891,
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"id": 3892,
"text": "Applying 50 μL IMAC wash solution, vortex and centrifuge at low speed (e.g., 800 rpm in a benchtop microcentrifuge) at room temperature to pellet the beads.",
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"id": 3564,
"text": "Use a 5× volume of 0.1 M glycine, pH 2.5.",
"checked": false,
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"id": 3567,
"text": "To enhance recovery, wash the matrix with an additional 5× volume of 0.1 M glycine, pH 2.5.",
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"id": 3568,
"text": "Adjust eluate to neutral pH by addition of an equal volume of 200 mM NH4HCO3.",
"checked": false,
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"checklist_items": [
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"id": 3569,
"text": "Add DTT to a final concentration of 10 mM or TCEP to a final concentration of 5 mM.",
"checked": false,
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"position": 0
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"id": 3570,
"text": "Incubate sample at room temperature with vortexing for 10 min with TCEP or 30 min with DTT.",
"checked": false,
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"position": 1
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{
"id": 3571,
"text": "Next, to alkylate free cysteines, add iodoacetamide to a final concentration of 10 mM",
"checked": false,
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"created_at": "2018-11-27T17:42:41.923Z",
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{
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"text": "Place at room temperature in the dark for 30 min.",
"checked": false,
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"created_at": "2018-11-27T17:42:41.934Z",
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"step": {
"id": 4874,
"name": "Digestion of the protein sample",
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"checklist_items": [
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"text": "Use an appropriate enzyme or chemical. If using trypsin, add sufficient enzyme for final trypsin: protein ratio of 1:20 to 1:100 (w/w).",
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"created_at": "2018-12-20T14:41:18.231Z",
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"text": "Seal the tube with Paraffin.",
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"text": "Incubate with end-over-end rotation at 37°C for 4 to 18 hours.",
"checked": false,
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"text": "Wash the column three times with 200 μl 100% acetonitrile.",
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},
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},
{
"step": {
"id": 4878,
"name": "Rinse column",
"description": "<!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\">\n<html><body><div>\n<strong>Three to ten times with 200 μL 0.1% TFA</strong>. The volume required for sufficient rinsing depends on the concentration of salts and other reagents in the sample. If electrospray/nanospray is to be performed, it is advisable to subsequently rinse the column three times with <strong>200 μL</strong> water to remove the TFA, which may interfere with ionization in electrospray/nanospray.</div></body></html>",
"position": 2,
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},
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{
"step": {
"id": 4877,
"name": "Application of 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>Follow guidelines for sample preparation. </p></body></html>",
"position": 1,
"completed": false,
"completed_on": null,
"user_id": 202,
"created_at": "2018-11-27T17:59:44.389Z",
"updated_at": "2018-12-24T08:07:53.994Z",
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},
"checklists": [
{
"checklist": {
"id": 861,
"name": "Preparation of samples:",
"step_id": 4877,
"created_at": "2018-11-27T17:59:44.392Z",
"updated_at": "2018-11-27T17:59:44.392Z",
"created_by_id": null,
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},
"checklist_items": [
{
"id": 3577,
"text": "Add 10% TFA to bring the final concentration to 0.1% TFA to the peptide sample.",
"checked": false,
"checklist_id": 861,
"created_at": "2018-11-27T17:59:44.394Z",
"updated_at": "2018-11-27T17:59:44.394Z",
"created_by_id": null,
"last_modified_by_id": null,
"position": 0
},
{
"id": 3578,
"text": "Apply the sample to the column",
"checked": false,
"checklist_id": 861,
"created_at": "2018-11-27T17:59:44.403Z",
"updated_at": "2018-11-27T17:59:44.403Z",
"created_by_id": null,
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"position": 1
},
{
"id": 3579,
"text": "Use a pipette to push sample through slowly",
"checked": false,
"checklist_id": 861,
"created_at": "2018-11-27T17:59:44.410Z",
"updated_at": "2018-11-27T17:59:44.410Z",
"created_by_id": null,
"last_modified_by_id": null,
"position": 2
}
]
}
],
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}
]
}
],
"results": []
}
],
"my_module_groups": [
{
"id": 1179,
"created_at": "2018-12-19T09:02:54.877Z",
"updated_at": "2018-12-19T09:02:54.877Z",
"created_by_id": 202,
"experiment_id": 454
}
]
}
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