scinote-web/app/assets/templates/experiment_497/experiment.json

{"experiment":{"id":497,"name":"SDS-PAGE for Protein Characterization","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.","project_id":223,"created_by_id":202,"last_modified_by_id":202,"archived":false,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-12-24T10:56:55.416Z","updated_at":"2019-01-28T15:15:24.331Z","workflowimg_file_name":"wimg20181224-1-1hyikrd.png","workflowimg_content_type":"image/png","workflowimg_file_size":2244,"workflowimg_updated_at":"2019-01-28T15:15:24.331Z","uuid":"8aefcdf3-ddb3-4174-8846-129a420426aa"},"my_modules":[{"my_module":{"id":2385,"name":"Preparation of polyacrylamide gel","due_date":null,"description":null,"x":0,"y":7,"my_module_group_id":1195,"created_at":"2018-12-24T11:00:55.170Z","updated_at":"2018-12-24T13:43:31.287Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":0,"experiment_id":497,"state":"uncompleted","completed_on":null},"outputs":[{"id":6050,"input_id":2384,"output_id":2385}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3902,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2385,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-12-24T11:00:55.177Z","updated_at":"2019-01-29T10:04:08.808Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5250,"name":"Make the stacking gel","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003eProteins 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.\u003cstrong\u003e\u003cbr\u003e\u003cbr\u003e5mL stacking gel:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003eH2O \u003cstrong\u003e2.975 mL\u003c/strong\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e0.5 M\u003c/strong\u003e Tris-HCl, \u003cstrong\u003epH 6.8 1.25 mL\u003c/strong\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e10%\u003c/strong\u003e (w/v) SDS \u003cstrong\u003e0.05 mL\u003c/strong\u003e\n\u003c/li\u003e\n\u003cli\u003eAcrylamide/Bis-acrylamide (\u003cstrong\u003e30%/0.8% w/v\u003c/strong\u003e) \u003cstrong\u003e0.67 mL\u003c/strong\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e10%\u003c/strong\u003e (w/v) ammonium persulfate (AP) \u003cstrong\u003e0.05 mL\u003c/strong\u003e\n\u003c/li\u003e\n\u003cli\u003eTEMED \u003cstrong\u003e0.005 mL\u003c/strong\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003cstrong\u003e\u003cstrong\u003e1x Running buffer:\u003cbr\u003e\u003c/strong\u003e\u003c/strong\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cstrong\u003e25 mM\u003c/strong\u003e Tris-HCl\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e200 mM\u003c/strong\u003e Glycine\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e0.1%\u003c/strong\u003e (w/v) SDS\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T11:30:55.051Z","updated_at":"2019-01-29T10:04:08.749Z","last_modified_by_id":202,"protocol_id":3902},"checklists":[{"checklist":{"id":973,"name":"Guideline","step_id":5250,"created_at":"2018-12-24T11:30:55.053Z","updated_at":"2018-12-24T11:30:55.053Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":4007,"text":"Discard the water and you can see separating gel left.","checked":false,"checklist_id":973,"created_at":"2018-12-24T11:30:55.054Z","updated_at":"2018-12-24T11:30:55.054Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":4008,"text":"Pipette in stacking gel until an overflow.","checked":false,"checklist_id":973,"created_at":"2018-12-24T11:30:55.060Z","updated_at":"2018-12-24T11:30:55.060Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":4009,"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,"created_at":"2018-12-24T11:30:55.065Z","updated_at":"2018-12-24T11:30:55.065Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":4010,"text":"Make sure a complete gelation of the stacking gel and take out the comb.","checked":false,"checklist_id":973,"created_at":"2018-12-24T11:30:55.071Z","updated_at":"2018-12-24T11:30:55.071Z","created_by_id":null,"last_modified_by_id":null,"position":3},{"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,"created_at":"2018-12-24T11:30:55.076Z","updated_at":"2018-12-24T11:30:55.076Z","created_by_id":null,"last_modified_by_id":null,"position":4},{"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,"created_at":"2018-12-24T11:30:55.081Z","updated_at":"2018-12-24T11:30:55.081Z","created_by_id":null,"last_modified_by_id":null,"position":5}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5248,"name":"Preparation of the equipment","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cdiv style=\"text-align: justify;\"\u003eGather combs, glass plates, spacer (silicone tubing), and binder clips. \u003cem\u003e\u003cstrong\u003eA comb is used to make wells (lanes) to load samples.\u003c/strong\u003e\u003c/em\u003e Use an appropriate comb depending on the sample size. Thoroughly clean the glass plates with ethanol, and assemble the gel casting mold, taking care to not leave any space.\u003c/div\u003e\n\u003cdiv style=\"text-align: justify;\"\u003e\n\u003cbr\u003e\u003cstrong\u003eExample:\u003c/strong\u003e Use an \u003cstrong\u003e8-lane\u003c/strong\u003e comb for \u003cstrong\u003e7 samples\u003c/strong\u003e and molecular weight markers.\u003c/div\u003e\n\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T11:04:34.946Z","updated_at":"2019-01-29T10:02:37.258Z","last_modified_by_id":202,"protocol_id":3902},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5249,"name":"Make the separating gel","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eThe acrylamide percentage in SDS PAGE gel depends on the size of the target protein in the sample. Using a higher acrylamide concentration produces a gel with a smaller mesh size suitable for the separation of small proteins. \u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T11:12:28.609Z","updated_at":"2019-01-29T10:02:55.233Z","last_modified_by_id":202,"protocol_id":3902},"checklists":[{"checklist":{"id":972,"name":"Guideline:","step_id":5249,"created_at":"2018-12-24T11:23:58.433Z","updated_at":"2018-12-24T11:23:58.433Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":4002,"text":"Prepare the gel solution in a small beaker. AP and TEMED must be added right before each use.","checked":false,"checklist_id":972,"created_at":"2018-12-24T11:23:58.434Z","updated_at":"2018-12-24T11:23:58.434Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":4003,"text":"Swirl the solution gently but thoroughly.","checked":false,"checklist_id":972,"created_at":"2018-12-24T11:23:58.442Z","updated_at":"2018-12-24T11:23:58.442Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":4004,"text":"Pipette appropriate amount of separating gel solution (listed above) into the gap between the glass plates.","checked":false,"checklist_id":972,"created_at":"2018-12-24T11:23:58.448Z","updated_at":"2018-12-24T11:23:58.448Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":4005,"text":"To make the top of the separating gel be horizontal, fill in water (either isopropanol) into the gap until a overflow. Water also prevents contact with oxygen, which inhibits polymerization.","checked":false,"checklist_id":972,"created_at":"2018-12-24T11:23:58.453Z","updated_at":"2018-12-24T11:26:09.996Z","created_by_id":null,"last_modified_by_id":null,"position":3},{"id":4006,"text":"Wait for 20-30min to let it gelate.","checked":false,"checklist_id":972,"created_at":"2018-12-24T11:23:58.459Z","updated_at":"2018-12-24T11:23:58.459Z","created_by_id":null,"last_modified_by_id":null,"position":4}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[{"id":548,"step_id":5249,"table_id":685}],"tables":[{"id":685,"created_at":"2018-12-24T11:20:43.674Z","updated_at":"2019-01-29T10:02:55.216Z","created_by_id":202,"last_modified_by_id":202,"name":"For 10 mL separating gel:","team_id":1,"contents":"eyJkYXRhIjpbWyJBY3lsYW1pZGUgcGVyY2VudGFnZSIsIjYlIiwiOCUiLCIx\nMCUiLCIxMiUiLCIxNSUiXSxbIkgyTyAobUwpIiwiNS4yIiwiNC42IiwiMy44\nIiwiMy4yIiwiMi4yIl0sWyJBY3J5bGFtaWRlL0Jpcy1hY3J5bGFtaWRlICAo\nbUwpIiwiMiIsIjIuNiIsIjMuNCIsIjQiLCI1Il0sWyIxLjVNIFRyaXM7IHBI\nPTguOCAobUwpIiwiMi42IiwiMi42IiwiMi42IiwiMi42IiwiMi42Il0sWyIx\nMCUgKHcvdilTRFMgKG1MKSIsIjAuMSIsIjAuMSIsIjAuMSIsIjAuMSIsIjAu\nMSJdLFsiMTAlICh3L3YpIGFtbW9uaXVtIHBlcnN1bGZhdGUgKEFQKSAozrxs\nKSIsIjEwMCIsIjEwMCIsIjEwMCIsIjEwMCIsIjEwMCJdLFsiVEVNRUQgKM68\nbCkiLCIxMCIsIjEwIiwiMTAiLCIxMCIsIjEwIl1dfQ==\n","data_vector":"JzAuMSc6MzcsMzgsMzksNDAsNDEgJzEuNSc6MjIgJzEwJzo1LDMzLDQyLDU3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\nJ3cvdic6MzQsNDM=\n"}]}]}],"results":[]},{"my_module":{"id":2384,"name":"Sample preparation","due_date":null,"description":null,"x":33,"y":7,"my_module_group_id":1195,"created_at":"2018-12-24T10:57:17.160Z","updated_at":"2018-12-24T13:43:31.290Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":1,"experiment_id":497,"state":"uncompleted","completed_on":null},"outputs":[{"id":6047,"input_id":2386,"output_id":2384}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3901,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2384,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-12-24T10:57:17.170Z","updated_at":"2019-01-29T10:05:04.164Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5251,"name":"Mix samples with buffer","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003eMake sure your target protein is \u003cstrong\u003edissolved in the liquid phase\u003c/strong\u003e, and \u003cstrong\u003eno inappropriate ingredients are present\u003c/strong\u003e (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.\u003cbr\u003e\u003cbr\u003e\u003cbr\u003eMix samples with loading buffer. Mix by flicking the tube.\u003cstrong\u003e\u003cbr\u003e\u003cbr\u003e5X Sample buffer (Loading buffer):\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cstrong\u003e10%\u003c/strong\u003e w/v SDS\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e10 mM\u003c/strong\u003e Dithiothreitol, or beta-mercapto-ethanol\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e20 %\u003c/strong\u003e v/v Glycerol\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e0.2 M\u003c/strong\u003e Tris-HCl; \u003cstrong\u003epH 6.8\u003c/strong\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e0.05%\u003c/strong\u003e w/v Bromophenolblue\u003c/li\u003e\n\u003c/ul\u003e\n\u003cstrong\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003c/strong\u003e\n\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T11:44:32.382Z","updated_at":"2019-01-29T10:04:52.137Z","last_modified_by_id":202,"protocol_id":3901},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5253,"name":"Centrifugation","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cstrong\u003eCentrifuge:\u003c/strong\u003e\u003cbr\u003e\u003cbr\u003e\n\u003cul\u003e\n\u003cli\u003e15,000 rpm\u003c/li\u003e\n\u003cli\u003e1 minute\u003c/li\u003e\n\u003cli\u003e4°C\u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003eUse \u003cstrong\u003ethe supernatant\u003c/strong\u003e for SDS-PAGE.\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T11:48:19.756Z","updated_at":"2018-12-24T11:48:19.756Z","last_modified_by_id":202,"protocol_id":3901},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5252,"name":"Heat the samples","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eHeat the samples at \u003cstrong\u003e100°C\u003c/strong\u003e for \u003cstrong\u003e3 minutes\u003c/strong\u003e in a heat block.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T11:47:05.158Z","updated_at":"2019-01-29T10:05:04.115Z","last_modified_by_id":202,"protocol_id":3901},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2386,"name":"Electrophoresis","due_date":null,"description":null,"x":66,"y":7,"my_module_group_id":1195,"created_at":"2018-12-24T12:27:59.858Z","updated_at":"2018-12-24T13:43:31.284Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":2,"experiment_id":497,"state":"uncompleted","completed_on":null},"outputs":[{"id":6048,"input_id":2387,"output_id":2386},{"id":6049,"input_id":2388,"output_id":2386}],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3903,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2386,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-12-24T12:27:59.867Z","updated_at":"2019-01-29T10:05:49.860Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5254,"name":"Sample loading","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eRemove air bubbles and small pieces of gel from the wells and under the gel using a syringe.\u003cbr\u003eLoad prepared samples into wells and make sure not to overflow. Don't forget loading \u003cstrong\u003eprotein marker into the first lane\u003c/strong\u003e. Then cover the top and connect the anodes.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:31:41.933Z","updated_at":"2019-01-28T15:33:39.705Z","last_modified_by_id":202,"protocol_id":3903},"checklists":[],"step_comments":[],"step_assets":[{"id":2973,"step_id":5254,"asset_id":3624}],"assets":[{"id":3624,"created_at":"2019-01-28T15:33:28.689Z","updated_at":"2019-01-28T15:33:39.696Z","file_file_name":"SDS_page_electrophoresis.PNG","file_content_type":"image/png","file_file_size":64782,"file_updated_at":"2019-01-28T15:33:38.768Z","created_by_id":202,"last_modified_by_id":null,"estimated_size":71260,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]},{"step":{"id":5256,"name":"Remove the gel","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eRemove the gel assembly from the electrophoresis apparatus. Remove the gel from the glass plates using a spatula, and prepare for subsequent analysis.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:39:15.168Z","updated_at":"2018-12-24T12:39:15.168Z","last_modified_by_id":202,"protocol_id":3903},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5255,"name":"Electrophoresis","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eSet an appropriate voltage and run the electrophoresis when everything's done.\u003cbr\u003e\u003cbr\u003eAs for the \u003cstrong\u003etotal running time\u003c/strong\u003e, stop SDS-PAGE running when the downmost sign of the protein marker (if no visible sign, inquire the manufacturer) almost reaches the foot line of the glass plate. Generally, about \u003cstrong\u003e1 hour\u003c/strong\u003e for a \u003cstrong\u003e120V\u003c/strong\u003e voltage and a \u003cstrong\u003e12%\u003c/strong\u003e separating gel. For a separating gel possessing a higher percentage of acylamide, the time will be longer.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:38:33.920Z","updated_at":"2019-01-29T10:05:49.827Z","last_modified_by_id":202,"protocol_id":3903},"checklists":[],"step_comments":[],"step_assets":[{"id":2974,"step_id":5255,"asset_id":3625}],"assets":[{"id":3625,"created_at":"2019-01-28T15:35:22.040Z","updated_at":"2019-01-29T10:05:49.772Z","file_file_name":"Separation_of_protein_mixture.PNG","file_content_type":"image/png","file_file_size":271493,"file_updated_at":"2019-01-28T15:35:39.862Z","created_by_id":202,"last_modified_by_id":202,"estimated_size":298642,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2388,"name":"Western Blot","due_date":null,"description":null,"x":99,"y":0,"my_module_group_id":1195,"created_at":"2018-12-24T13:08:01.834Z","updated_at":"2018-12-24T13:43:31.275Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":3,"experiment_id":497,"state":"uncompleted","completed_on":null},"outputs":[],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3905,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2388,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-12-24T13:08:01.843Z","updated_at":"2019-01-31T09:18:18.471Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5262,"name":"Transfer of proteins to a membrane","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003eSoak a piece of \u003cstrong\u003ePVDF membrane\u003c/strong\u003e slightly larger than the gel in methanol for \u003cstrong\u003e1 minute\u003c/strong\u003e (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.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eTransfer buffer:\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cstrong\u003e25 mM\u003c/strong\u003e Tris-HCl\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e192 mM\u003c/strong\u003e glycine\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e20%\u003c/strong\u003e MeOH\u003c/li\u003e\n\u003c/ul\u003e\n\u003cbr\u003eEquilibrate the gel in transfer buffer after electrophoresis. \u003cbr\u003e\u003cbr\u003ePlace 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.\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T13:17:10.122Z","updated_at":"2019-01-31T09:18:18.436Z","last_modified_by_id":202,"protocol_id":3905},"checklists":[],"step_comments":[],"step_assets":[{"id":2975,"step_id":5262,"asset_id":3626}],"assets":[{"id":3626,"created_at":"2019-01-28T15:48:11.052Z","updated_at":"2019-01-29T10:06:20.918Z","file_file_name":"Western_Blot_transfer.PNG","file_content_type":"image/png","file_file_size":3029,"file_updated_at":"2019-01-28T15:48:42.879Z","created_by_id":202,"last_modified_by_id":202,"estimated_size":3331,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]},{"step":{"id":5263,"name":"Blotting","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003eConnect \u003cstrong\u003ethe plus electrode to the anode plate\u003c/strong\u003e and connect the \u003cstrong\u003enegative electrode to the cathode plate\u003c/strong\u003e.\u003cbr\u003eTurn on the power supply and transfer for \u003cstrong\u003e1 hour\u003c/strong\u003e at \u003cstrong\u003e50 mA\u003c/strong\u003e (for one gel).\u003cbr\u003e\u003cbr\u003eTurn 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.\u003cbr\u003e\u003cstrong\u003ePonceau S\u003c/strong\u003e is commonly used for this purpose because the membrane is easily destained, and it does not interfere with subsequent probing with antibodies.\u003cbr\u003e\u003cbr\u003e\u003cstrong\u003ePonceau S solution: \u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cstrong\u003e1g\u003c/strong\u003e Ponceau S\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e50mL\u003c/strong\u003e acetic acid\u003c/li\u003e\n\u003cli\u003eMake up to \u003cstrong\u003e1L\u003c/strong\u003e with ddH2O\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T13:24:09.108Z","updated_at":"2019-01-29T10:07:20.397Z","last_modified_by_id":202,"protocol_id":3905},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5264,"name":"Probing with antibodies and detection of the bands","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cp\u003e\u003cstrong\u003eBlocking buffer: \u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n\u003cli\u003e\n\u003cstrong\u003e1-3% BSA in PBS-T: \u003c/strong\u003eBovine serum albumin (BSA) is used as a blocking agent.\u003c/li\u003e\n\u003cli\u003e\n\u003cstrong\u003e1-5% skim milk in PBS-T \u003c/strong\u003eSkim milk is more effective in blocking and used frequently. However, it can interfere with specific antigen-antibody reactions. \u003cem\u003e\u003cstrong\u003eSkim milk contains the \u003c/strong\u003e\u003cstrong\u003ephosphoprotein \u003c/strong\u003e\u003cstrong\u003ecasein and therefore is not suitable for the detection of phosphoproteins.\u003c/strong\u003e\u003c/em\u003e\n\u003c/li\u003e\n\u003c/ul\u003e\n\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T13:33:23.306Z","updated_at":"2018-12-24T13:33:53.668Z","last_modified_by_id":202,"protocol_id":3905},"checklists":[{"checklist":{"id":974,"name":"Guidelines","step_id":5264,"created_at":"2018-12-24T13:33:23.308Z","updated_at":"2018-12-24T13:33:23.308Z","created_by_id":null,"last_modified_by_id":null},"checklist_items":[{"id":4013,"text":"Place the membrane in blocking buffer and incubate at room temperature for 1 hour (or at 4°C overnight) with shaking.","checked":false,"checklist_id":974,"created_at":"2018-12-24T13:33:23.310Z","updated_at":"2018-12-24T13:33:23.310Z","created_by_id":null,"last_modified_by_id":null,"position":0},{"id":4014,"text":"Wash the membrane with washing buffer 3 times (5 minutes each time).","checked":false,"checklist_id":974,"created_at":"2018-12-24T13:33:23.316Z","updated_at":"2018-12-24T13:33:23.316Z","created_by_id":null,"last_modified_by_id":null,"position":1},{"id":4015,"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.","checked":false,"checklist_id":974,"created_at":"2018-12-24T13:33:23.321Z","updated_at":"2018-12-24T13:33:23.321Z","created_by_id":null,"last_modified_by_id":null,"position":2},{"id":4016,"text":"Wash the membrane with washing buffer 3 times (10 minutes each time).","checked":false,"checklist_id":974,"created_at":"2018-12-24T13:33:23.327Z","updated_at":"2018-12-24T13:33:23.327Z","created_by_id":null,"last_modified_by_id":null,"position":3},{"id":4017,"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.","checked":false,"checklist_id":974,"created_at":"2018-12-24T13:33:23.332Z","updated_at":"2018-12-24T13:33:23.332Z","created_by_id":null,"last_modified_by_id":null,"position":4},{"id":4018,"text":"Wash the membrane with washing buffer 3 times (10 minutes each time).","checked":false,"checklist_id":974,"created_at":"2018-12-24T13:33:23.337Z","updated_at":"2018-12-24T13:33:23.337Z","created_by_id":null,"last_modified_by_id":null,"position":5}]}],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5265,"name":"Chemiluminescence method","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003ePrepare 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 \u003cstrong\u003e1 minute\u003c/strong\u003e, and remove the reagent using a pipette. \u003cbr\u003eWrap 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.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":3,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T13:34:41.460Z","updated_at":"2019-01-29T10:07:43.690Z","last_modified_by_id":202,"protocol_id":3905},"checklists":[],"step_comments":[],"step_assets":[{"id":2976,"step_id":5265,"asset_id":3627}],"assets":[{"id":3627,"created_at":"2019-01-28T15:50:42.310Z","updated_at":"2019-01-29T10:07:43.634Z","file_file_name":"Detection.PNG","file_content_type":"image/png","file_file_size":10299,"file_updated_at":"2019-01-28T15:50:43.265Z","created_by_id":202,"last_modified_by_id":202,"estimated_size":11328,"file_present":true,"lock":null,"lock_ttl":null,"version":1,"file_processing":false,"team_id":1}],"step_tables":[],"tables":[]}]}],"results":[]},{"my_module":{"id":2387,"name":"Standard Coomassie Stain","due_date":null,"description":null,"x":99,"y":14,"my_module_group_id":1195,"created_at":"2018-12-24T12:42:30.757Z","updated_at":"2018-12-24T13:43:31.281Z","archived":false,"archived_on":null,"created_by_id":202,"last_modified_by_id":202,"archived_by_id":null,"restored_by_id":null,"restored_on":null,"nr_of_assigned_samples":0,"workflow_order":4,"experiment_id":497,"state":"uncompleted","completed_on":null},"outputs":[],"my_module_tags":[],"task_comments":[],"my_module_repository_rows":[],"user_my_modules":[],"protocols":[{"protocol":{"id":3904,"name":null,"authors":null,"description":null,"added_by_id":null,"my_module_id":2387,"team_id":1,"protocol_type":"unlinked","parent_id":null,"parent_updated_at":null,"archived_by_id":null,"archived_on":null,"restored_by_id":null,"restored_on":null,"created_at":"2018-12-24T12:42:30.765Z","updated_at":"2019-01-29T10:21:07.111Z","published_on":null,"nr_of_linked_children":0},"protocol_protocol_keywords":[],"steps":[{"step":{"id":5260,"name":"Destain","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cstrong\u003eDestain solution\u003c/strong\u003e: Add \u003cstrong\u003e500mL\u003c/strong\u003e of HPLC- grade methanol to \u003cstrong\u003e300 mL\u003c/strong\u003e of HPLC grade water. Add \u003cstrong\u003e100 mL\u003c/strong\u003e of reagent grade acetic acid and, after mixing, adjust the total volume to \u003cstrong\u003e1000mL\u003c/strong\u003e with water. The final concentrations are \u003cstrong\u003e50%\u003c/strong\u003e (v/v) methanol in water with \u003cstrong\u003e10%\u003c/strong\u003e (v/v) acetic acid. \u003cbr\u003e\u003cbr\u003eCover the gel with\u003cstrong\u003e ~250mL\u003c/strong\u003e 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.\u003c/body\u003e\u003c/html\u003e","position":3,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:50:15.910Z","updated_at":"2019-01-29T10:20:35.719Z","last_modified_by_id":202,"protocol_id":3904},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5258,"name":"Washing of the gel","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003eCover the gel with \u003cstrong\u003e500mL\u003c/strong\u003e of the gel-washing solution.  \u003cbr\u003e\u003cbr\u003e\u003cstrong\u003eGel-washing solution:\u003c/strong\u003e Add \u003cstrong\u003e500mL\u003c/strong\u003e of HPLC-grade methanol to \u003cstrong\u003e300 mL\u003c/strong\u003e of HPLC grade water. Add \u003cstrong\u003e100mL\u003c/strong\u003e of reagent grade acetic acid and adjust the total volume to \u003cstrong\u003e1000 mL\u003c/strong\u003e with HPLC grade water. The final concentrations are \u003cstrong\u003e50%\u003c/strong\u003e (v/v) methanol in water with \u003cstrong\u003e10%\u003c/strong\u003e (v/v) acetic acid. \u003cbr\u003e\u003cbr\u003eContinue 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.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":1,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:47:54.498Z","updated_at":"2019-01-29T10:09:33.373Z","last_modified_by_id":202,"protocol_id":3904},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5259,"name":"Comassie staining","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\u003cp\u003e\u003cstrong\u003eComassie Stain:\u003c/strong\u003e Dissolve \u003cstrong\u003e0.4g\u003c/strong\u003e of Coomassie blue R350 in \u003cstrong\u003e200 mL\u003c/strong\u003e of \u003cstrong\u003e40%\u003c/strong\u003e (v/v) HPLC grade methanol in water with stirring as needed. Filter the solution to remove any insoluble material. Add \u003cstrong\u003e200mL\u003c/strong\u003e of \u003cstrong\u003e20%\u003c/strong\u003e (v/v) acetic acid in water. The final concentration is \u003cstrong\u003e0.1%\u003c/strong\u003e (w/v) Coomassie blue R350, \u003cstrong\u003e20%\u003c/strong\u003e (v/v) methanol, and \u003cstrong\u003e10%\u003c/strong\u003e (v/v) acetic acid.\u003cbr\u003e\u003cbr\u003eCover the gel with \u003cstrong\u003e400mL\u003c/strong\u003e of the Coomassie stain. Stain the gel at room temperature for \u003cstrong\u003e3 to 4 hours\u003c/strong\u003e with gentle agitation. The Coomassie stain is removed by aspiration after staining.\u003c/p\u003e\u003c/body\u003e\u003c/html\u003e","position":2,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:49:39.311Z","updated_at":"2019-01-29T10:19:56.594Z","last_modified_by_id":202,"protocol_id":3904},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5261,"name":"Storage of gels","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cstrong\u003eStorage solution:\u003c/strong\u003e Add \u003cstrong\u003e25mL\u003c/strong\u003e of reagent grade acetic acid to \u003cstrong\u003e400mL\u003c/strong\u003e of HPLC grade water. After mixing, adjust the final volume to \u003cstrong\u003e500mL\u003c/strong\u003e with water. The final concentration of acetic acid is \u003cstrong\u003e5%\u003c/strong\u003e (v/v).\u003cbr\u003e\u003cbr\u003eEquilibrate the gel in the \u003cstrong\u003e500mL\u003c/strong\u003e of the storage solution for at least \u003cstrong\u003e1 hour\u003c/strong\u003e. The gel should return to its original dimensions during this process. Store the gel in the storage solution as needed. It might be convenient to carefully transfer the gel to a heat-sealable bag for longer-term storage.\u003c/body\u003e\u003c/html\u003e","position":4,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:52:54.562Z","updated_at":"2019-01-29T10:21:07.061Z","last_modified_by_id":202,"protocol_id":3904},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]},{"step":{"id":5257,"name":"Gel fixation","description":"\u003c!DOCTYPE html PUBLIC \"-//W3C//DTD HTML 4.0 Transitional//EN\" \"http://www.w3.org/TR/REC-html40/loose.dtd\"\u003e\n\u003chtml\u003e\u003cbody\u003e\n\u003cstrong\u003eGel-fixing solution:\u003c/strong\u003e Add \u003cstrong\u003e500mL\u003c/strong\u003e of USP-grade \u003cstrong\u003e95% (v/v)\u003c/strong\u003e ethanol to \u003cstrong\u003e300 mL\u003c/strong\u003e of HPLC grade water. Add \u003cstrong\u003e100 mL\u003c/strong\u003e of reagent grade acetic acid and adjust the total volume to \u003cstrong\u003e1000 mL\u003c/strong\u003e with water. The final concentrations are \u003cstrong\u003e50%\u003c/strong\u003e (v/v) ethanol in water with \u003cstrong\u003e10%\u003c/strong\u003e (v/v) acetic acid.\u003cbr\u003e\u003cbr\u003eAfter electrophoresis, the gel is washed off the glass plates with 500 mL of the gel-fixing solution and soaked in that solution for \u003cstrong\u003e1 hour.\u003c/strong\u003e\u003cbr\u003e\u003cbr\u003eThe purpose of this step is to gently remove the gel from the plate and begin washing the SDS-containing gel buffers out of the gel. At the end of this time, remove the solution by aspiration.\u003c/body\u003e\u003c/html\u003e","position":0,"completed":false,"completed_on":null,"user_id":202,"created_at":"2018-12-24T12:45:54.303Z","updated_at":"2019-01-29T10:08:50.637Z","last_modified_by_id":202,"protocol_id":3904},"checklists":[],"step_comments":[],"step_assets":[],"assets":[],"step_tables":[],"tables":[]}]}],"results":[]}],"my_module_groups":[{"id":1195,"created_at":"2018-12-24T13:43:31.272Z","updated_at":"2018-12-24T13:43:31.272Z","created_by_id":202,"experiment_id":497}]}