{ "experiment": { "id": 423, "name": "Antibody Purification", "description": "Antibody purification involves selective enrichment or specific isolation of antibodies from serum, ascites fluid or cell culture supernatant of a hybridoma cell line. Purification methods range from very crude to highly specific.\r\nOnce they are purified (and possibly after labelling them with an enzyme or fluorescent tag), these antibodies can be used directly to probe the specific antigen in Western blotting, ELISA and other applications.", "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": "2018-11-08T10:28:48.389Z", "updated_at": "2018-12-21T13:26:16.207Z", "uuid": "37198411-2ad0-4198-b680-47f3b455ce0d" }, "my_modules": [ { "my_module": { "id": 2097, "name": "Polishing", "due_date": null, "description": null, "x": 72, "y": 0, "my_module_group_id": 1190, "created_at": "2018-11-08T10:28:49.322Z", "updated_at": "2018-12-21T13:25:20.391Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": null, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 2, "experiment_id": 423, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 6024, "input_id": 2098, "output_id": 2097 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3407, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2097, "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": "2018-11-08T10:28:49.390Z", "updated_at": "2019-02-20T07:39:44.494Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4287, "name": "Before applying a new sample", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-08T10:28:49.408Z", "updated_at": "2018-12-21T13:31:50.804Z", "last_modified_by_id": 202, "protocol_id": 3407 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n
Re-equilibrate column with buffer until the baseline of the monitored signal is stable. Proteins can be monitored at 280 nm.
" }, "position": 0 } ] }, { "step": { "id": 4290, "name": "Polishing", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-08T10:28:49.496Z", "updated_at": "2019-02-07T10:46:31.811Z", "last_modified_by_id": 202, "protocol_id": 3407 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\nEquilibrate with 2 column volumes of the buffer at a recommended flow rate (See the table). Apply a sample volume equivalent to between 0.3% and 4% of the bed volume. For most applications, the sample volume should not exceed 2% to achieve high resolution. Smaller sample volumes generally lead to an improved resolution.
Elute with 1 column volume of buffer solution (10 to 50 mM sodium phosphate, 150 mM sodium chloride, pH 7.0 to 7.4, or select the buffer in which the sample should be stored or solubilized for the next step). The sample should be fully dissolved. Centrifuge or filter to remove particulate matter.
Follow the preparation guidelines for capture task.
" }, "position": 0 }, { "checklist": { "checklist": { "id": 968, "name": "Guideline:", "step_id": 4286, "created_at": "2018-12-21T13:27:09.411Z", "updated_at": "2018-12-21T13:27:09.411Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3984, "text": "Equilibrate all materials to the temperature at which the separation will be performed.", "checked": false, "checklist_id": 968, "created_at": "2018-12-21T13:27:09.413Z", "updated_at": "2018-12-21T13:27:09.413Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3985, "text": "Eliminate air by flushing column end pieces with buffer. Ensure no air is trapped under the column net. Close column outlet leaving 1–2 cm of the buffer in the column.", "checked": false, "checklist_id": 968, "created_at": "2018-12-21T13:27:09.421Z", "updated_at": "2018-12-21T13:27:09.421Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3986, "text": "Gently resuspend the medium. For media not supplied in suspension, use a medium: buffer ratio of approximately 1:2 to produce a suspension for mixing during rehydration. Avoid using magnetic stirrers since they may damage the matrix.", "checked": false, "checklist_id": 968, "created_at": "2018-12-21T13:27:09.427Z", "updated_at": "2018-12-21T13:27:09.427Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 } ] }, "position": 1 } ] }, { "step": { "id": 4279, "name": "Stop the pump and close the column outlet.", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-08T10:28:49.069Z", "updated_at": "2018-12-21T13:29:08.411Z", "last_modified_by_id": 202, "protocol_id": 3405 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\nRemove the top piece and carefully fill the rest of the column with buffer to form an upward meniscus at the top. Insert the adaptor into the column at an angle, ensuring that no air is trapped under the net. Slide the adaptor slowly down the column (the outlet of the adaptor should be open) until the mark is reached. Lock the adaptor in position. Connect the column to the pump and begin equilibration. Re-position the adaptor if necessary.
" }, "position": 0 } ] }, { "step": { "id": 4283, "name": "Column fill and setting the column outlet.", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-08T10:28:49.179Z", "updated_at": "2019-02-19T14:49:30.685Z", "last_modified_by_id": 202, "protocol_id": 3405 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\nEstimate the amount of resuspended medium required and our the required volume of the medium into the column. Pouring down a glass rod held against the wall of the column will minimize the introduction of air bubbles. Immediately fill the column with buffer. Mount the column top piece and connect to a pump. Open the column outlet and set the pump to the desired flow rate. If the recommended flow rate cannot be obtained, use the maximum flow rate the pump can deliver. Do not exceed the maximum operating pressure of the medium or column. Maintain the packing flow rate for at least 3 column volumes after a constant bed height is obtained. Mark the bed height on the column. Do not exceed 75% of the packing flow rate during any purification.
" }, "position": 0 } ] } ] } ], "results": [ ] }, { "my_module": { "id": 2095, "name": "Buffer exchange and desalting", "due_date": null, "description": null, "x": 0, "y": 0, "my_module_group_id": 1190, "created_at": "2018-11-08T10:28:48.686Z", "updated_at": "2018-12-21T13:25:20.385Z", "archived": false, "archived_on": null, "created_by_id": 202, "last_modified_by_id": null, "archived_by_id": null, "restored_by_id": null, "restored_on": null, "workflow_order": 0, "experiment_id": 423, "state": "uncompleted", "completed_on": null }, "outputs": [ { "id": 6022, "input_id": 2096, "output_id": 2095 } ], "my_module_tags": [ ], "task_comments": [ ], "my_module_repository_rows": [ ], "user_my_modules": [ ], "protocols": [ { "protocol": { "id": 3403, "name": null, "authors": null, "description": null, "added_by_id": null, "my_module_id": 2095, "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": "2018-11-08T10:28:48.743Z", "updated_at": "2019-02-20T07:39:23.923Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 4273, "name": "Buffer exchange and desalting", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-11-08T10:28:48.761Z", "updated_at": "2019-02-20T07:39:28.712Z", "last_modified_by_id": 202, "protocol_id": 3403 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3753, "created_at": "2019-02-20T07:39:23.642Z", "updated_at": "2019-02-20T07:39:28.703Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 29576, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Desalting_and_Buffer_Exchange.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\nFill the syringe with buffer. To avoid introducing air into the column, connect the column \"drop to drop\" to the syringe (via the adapter). Wash the column. Use 25 mL buffer at 5 mL/min to remove completely the 20% ethanol (supplied as storage buffer). If air is trapped in the column, wash with degassed buffer until the air disappears. Air bubbles introduced onto the column by accident during sample application do not influence the separation.
Applying the sample. Use a 2–5 mL syringe at a flow rate between 1–10 mL/min. Discard the liquid eluted from the column. If the sample volume is less than 1.5 mL, change to buffer and proceed with the injection until a total of 1.5 mL has been eluted. Discard the eluted liquid.pter). Elute the protein with the appropriate volume.
Select volume from the table.
Follow guidelines for centrifuge set up.
" }, "position": 0 }, { "checklist": { "checklist": { "id": 934, "name": "Before use:", "step_id": 5209, "created_at": "2018-12-18T14:10:46.321Z", "updated_at": "2018-12-18T14:10:46.321Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3838, "text": "Check the inside of the centrifuge and the rotors to ensure that everything is dry.", "checked": false, "checklist_id": 934, "created_at": "2018-12-18T14:10:46.323Z", "updated_at": "2018-12-21T12:47:12.263Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3839, "text": "Check that shock-absorbing pads are in the bottom of the centrifuge buckets.", "checked": false, "checklist_id": 934, "created_at": "2018-12-18T14:10:46.331Z", "updated_at": "2018-12-18T14:10:46.331Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3840, "text": "Balance the opposing buckets by weighing them with their tubes on an open two-pan balance.", "checked": false, "checklist_id": 934, "created_at": "2018-12-18T14:10:46.337Z", "updated_at": "2018-12-18T14:10:46.337Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3841, "text": "Never fill centrifuge tubes to more than three-quarters capacity.", "checked": false, "checklist_id": 934, "created_at": "2018-12-18T14:10:46.344Z", "updated_at": "2018-12-18T14:10:46.344Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3842, "text": "Symmetrically distribute balanced tubes in opposing buckets. Always operate the centrifuge with all buckets in place, even if two opposing buckets are empty.", "checked": false, "checklist_id": 934, "created_at": "2018-12-18T14:10:46.354Z", "updated_at": "2018-12-18T14:10:46.354Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 } ] }, "position": 1 } ] }, { "step": { "id": 5210, "name": "Centrifugation of samples", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-18T14:12:05.603Z", "updated_at": "2019-02-07T10:41:20.533Z", "last_modified_by_id": 202, "protocol_id": 3403 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\nSwitch on and follow the manufacturer's instructions to set the centrifugation conditions.
For small sample volumes or proteins that adsorb non-specifically to filters, centrifuge at 10 000 x g for 15 minutes. For cell lysates, centrifuge at 40 000–50 000 x g for 30 minutes. Close and lock the lid and then start the centrifuge cycle. If excessive vibration occurs, or if a crack is heard or tube breakage is suspected, switch off the unit. Open the centrifuge only after the signal for the end of centrifugation is seen. Remove the sealed buckets (not tubes) slowly and carefully to prevent re-suspension.
Follow guidelines below to perform a Bradford assay.
" }, "position": 0 }, { "checklist": { "checklist": { "id": 969, "name": "Guideline:", "step_id": 4643, "created_at": "2018-12-21T13:35:09.660Z", "updated_at": "2018-12-21T13:35:09.660Z", "created_by_id": null, "last_modified_by_id": null }, "checklist_items": [ { "id": 3987, "text": "Prepare five to eight dilutions of BSA standard with a range of 5 to 100 µg protein.", "checked": false, "checklist_id": 969, "created_at": "2018-12-21T13:35:09.662Z", "updated_at": "2018-12-21T13:35:09.662Z", "created_by_id": null, "last_modified_by_id": null, "position": 0 }, { "id": 3988, "text": "Dilute protein samples to obtain 5-100 µg protein/30 µL.", "checked": false, "checklist_id": 969, "created_at": "2018-12-21T13:35:09.669Z", "updated_at": "2018-12-21T13:35:09.669Z", "created_by_id": null, "last_modified_by_id": null, "position": 1 }, { "id": 3989, "text": "Set two blank tubes. For the standard curve, add 30 µL of water instead of the standard solution. For the unknown protein samples, add 30 µL protein preparation buffer instead. Protein solutions are normally assayed in duplicate or triplicate.", "checked": false, "checklist_id": 969, "created_at": "2018-12-21T13:35:09.675Z", "updated_at": "2018-12-21T13:35:09.675Z", "created_by_id": null, "last_modified_by_id": null, "position": 2 }, { "id": 3990, "text": "Add 1.5 mL of Bradford reagent to each tube and mix well.", "checked": false, "checklist_id": 969, "created_at": "2018-12-21T13:35:09.682Z", "updated_at": "2018-12-21T13:35:09.682Z", "created_by_id": null, "last_modified_by_id": null, "position": 3 }, { "id": 3991, "text": "Incubate at room temperature for at least 5 min.", "checked": false, "checklist_id": 969, "created_at": "2018-12-21T13:35:09.688Z", "updated_at": "2018-12-21T13:35:09.688Z", "created_by_id": null, "last_modified_by_id": null, "position": 4 }, { "id": 3992, "text": "Absorbance will increase over time; samples should incubate at RT for no more than 1 h.", "checked": false, "checklist_id": 969, "created_at": "2018-12-21T13:35:09.703Z", "updated_at": "2018-12-21T13:35:09.703Z", "created_by_id": null, "last_modified_by_id": null, "position": 5 }, { "id": 3993, "text": "Measure absorbance at 595 nm.", "checked": false, "checklist_id": 969, "created_at": "2018-12-21T13:35:09.710Z", "updated_at": "2018-12-21T13:35:09.710Z", "created_by_id": null, "last_modified_by_id": null, "position": 6 } ] }, "position": 1 } ] } ] } ], "results": [ ] } ], "my_module_groups": [ { "id": 1190, "created_at": "2018-12-21T13:25:20.378Z", "updated_at": "2018-12-21T13:25:20.378Z", "created_by_id": 202, "experiment_id": 423 } ] }