{ "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", "uuid": "3363b358-1478-402c-8597-57739d3fb2e3" }, "my_modules": [ { "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, "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": "2019-02-20T07:31:00.167Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5264, "name": "Probing with antibodies and detection of the bands", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Blocking buffer: 

\n\n" }, "position": 0 }, { "checklist": { "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 } ] }, "position": 1 } ] }, { "step": { "id": 5262, "name": "Transfer of proteins to a membrane", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T13:17:10.122Z", "updated_at": "2019-02-20T07:30:20.884Z", "last_modified_by_id": 202, "protocol_id": 3905 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3736, "created_at": "2019-02-20T07:29:55.862Z", "updated_at": "2019-02-20T07:30:20.876Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 18346, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Western_Blot_Transfer.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Soak a piece of PVDF membrane slightly larger than the gel in methanol for 1 minute (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.

Transfer buffer:

\n\n
Equilibrate the gel in transfer buffer after electrophoresis. 

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." }, "position": 0 } ] }, { "step": { "id": 5263, "name": "Blotting", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T13:24:09.108Z", "updated_at": "2019-02-19T14:04:24.998Z", "last_modified_by_id": 202, "protocol_id": 3905 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Connect the plus electrode to the anode plate and connect the negative electrode to the cathode plate.
Turn on the power supply and transfer for 1 hour at 50 mA (for one gel).

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.
Ponceau S is commonly used for this purpose because the membrane is easily destained, and it does not interfere with subsequent probing with antibodies.

Ponceau S solution: 

\n\n" }, "position": 0 } ] }, { "step": { "id": 5265, "name": "Chemiluminescence method", "position": 3, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T13:34:41.460Z", "updated_at": "2019-02-20T07:31:21.254Z", "last_modified_by_id": 202, "protocol_id": 3905 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3737, "created_at": "2019-02-20T07:30:59.882Z", "updated_at": "2019-02-20T07:31:21.246Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 14173, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Detection.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n

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 1 minute, and remove the reagent using a pipette. 
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.

" }, "position": 0 } ] } ] } ], "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, "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": "2019-02-19T14:04:53.999Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5260, "name": "Destain", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nDestain solution: Add 500mL of HPLC- grade methanol to 300 mL of HPLC grade water. Add 100 mL of reagent grade acetic acid and, after mixing, adjust the total volume to 1000mL with water. The final concentrations are 50% (v/v) methanol in water with 10% (v/v) acetic acid.

Cover the gel with ~250mL 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." }, "position": 0 } ] }, { "step": { "id": 5258, "name": "Washing of the gel", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Cover the gel with 500mL of the gel-washing solution.  

Gel-washing solution: Add 500mL of HPLC-grade methanol to 300 mL of HPLC grade water. Add 100mL of reagent grade acetic acid and adjust the total volume to 1000 mL with HPLC grade water. The final concentrations are 50% (v/v) methanol in water with 10% (v/v) acetic acid.

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.

" }, "position": 0 } ] }, { "step": { "id": 5259, "name": "Comassie staining", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Comassie Stain: Dissolve 0.4g of Coomassie blue R350 in 200 mL of 40% (v/v) HPLC grade methanol in water with stirring as needed. Filter the solution to remove any insoluble material. Add 200mL of 20% (v/v) acetic acid in water. The final concentration is 0.1% (w/v) Coomassie blue R350, 20% (v/v) methanol, and 10% (v/v) acetic acid.

Cover the gel with 400mL of the Coomassie stain. Stain the gel at room temperature for 3 to 4 hours with gentle agitation. The Coomassie stain is removed by aspiration after staining.

" }, "position": 0 } ] }, { "step": { "id": 5257, "name": "Gel fixation", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T12:45:54.303Z", "updated_at": "2019-02-19T14:04:53.947Z", "last_modified_by_id": 202, "protocol_id": 3904 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nGel-fixing solution: Add 500mL of USP-grade 95% (v/v) ethanol to 300 mL of HPLC grade water. Add 100 mL of reagent grade acetic acid and adjust the total volume to 1000 mL with water. The final concentrations are 50% (v/v) ethanol in water with 10% (v/v) acetic acid.

After electrophoresis, the gel is washed off the glass plates with 500 mL of the gel-fixing solution and soaked in that solution for 1 hour.

The 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." }, "position": 0 } ] }, { "step": { "id": 5261, "name": "Storage of gels", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nStorage solution: Add 25mL of reagent grade acetic acid to 400mL of HPLC grade water. After mixing, adjust the final volume to 500mL with water. The final concentration of acetic acid is 5% (v/v).

Equilibrate the gel in the 500mL of the storage solution for at least 1 hour. 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." }, "position": 0 } ] } ] } ], "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, "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": "2019-02-20T07:29:26.888Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5254, "name": "Sample loading", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T12:31:41.933Z", "updated_at": "2019-02-20T07:28:19.933Z", "last_modified_by_id": 202, "protocol_id": 3903 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3734, "created_at": "2019-02-20T07:28:16.226Z", "updated_at": "2019-02-20T07:28:19.924Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 12542, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "SDS-PAGE_electrophoresis.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n

Remove air bubbles and small pieces of gel from the wells and under the gel using a syringe.
Load prepared samples into wells and make sure not to overflow. Don't forget loading protein marker into the first lane. Then cover the top and connect the anodes.

" }, "position": 0 } ] }, { "step": { "id": 5256, "name": "Remove the gel", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Remove the gel assembly from the electrophoresis apparatus. Remove the gel from the glass plates using a spatula, and prepare for subsequent analysis.

" }, "position": 0 } ] }, { "step": { "id": 5255, "name": "Electrophoresis", "position": 1, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T12:38:33.920Z", "updated_at": "2019-02-20T07:30:20.395Z", "last_modified_by_id": 202, "protocol_id": 3903 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3735, "created_at": "2019-02-20T07:29:26.573Z", "updated_at": "2019-02-20T07:30:20.386Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 19250, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Separation_of_protein_mixture.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n

Set an appropriate voltage and run the electrophoresis when everything's done.

As for the total running time, 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 1 hour for a 120V voltage and a 12% separating gel. For a separating gel possessing a higher percentage of acylamide, the time will be longer.

" }, "position": 0 } ] } ] } ], "results": [ ] }, { "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, "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": "2019-02-20T07:25:44.713Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5248, "name": "Preparation of the equipment", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n
Gather combs, glass plates, spacer (silicone tubing), and binder clips. A comb is used to make wells (lanes) to load samples. 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.
\n
\n
Example: Use an 8-lane comb for 7 samples and molecular weight markers.
\n" }, "position": 0 } ] }, { "step": { "id": 5249, "name": "Make the separating gel", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

The 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. 

" }, "position": 0 }, { "table": { "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" }, "position": 1 }, { "checklist": { "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 } ] }, "position": 2 } ] }, { "step": { "id": 5250, "name": "Make the stacking gel", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T11:30:55.051Z", "updated_at": "2019-02-20T07:26:19.503Z", "last_modified_by_id": 202, "protocol_id": 3902 }, "step_comments": [ ], "assets": [ { "asset": { "id": 3733, "created_at": "2019-02-20T07:25:44.445Z", "updated_at": "2019-02-20T07:26:19.494Z", "created_by_id": 202, "last_modified_by_id": null, "estimated_size": 48073, "lock": null, "lock_ttl": null, "version": 1, "file_processing": false, "team_id": 1 }, "asset_blob": { "filename": "Gel_Preparation.jpg" } } ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

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.

5mL stacking gel:

\n\n1x Running buffer:
\n\n" }, "position": 0 }, { "checklist": { "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 } ] }, "position": 1 } ] } ] } ], "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, "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": "2019-02-19T14:03:57.520Z", "published_on": null, "nr_of_linked_children": 0 }, "protocol_protocol_keywords": [ ], "steps": [ { "step": { "id": 5253, "name": "Centrifugation", "position": 2, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T11:48:19.756Z", "updated_at": "2019-02-19T14:03:57.459Z", "last_modified_by_id": 202, "protocol_id": 3901 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\nCentrifuge:

\n\n
Use the supernatant for SDS-PAGE." }, "position": 0 } ] }, { "step": { "id": 5252, "name": "Heat the samples", "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 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n

Heat the samples at 100°C for 3 minutes in a heat block.

" }, "position": 0 } ] }, { "step": { "id": 5251, "name": "Mix samples with buffer", "position": 0, "completed": false, "completed_on": null, "user_id": 202, "created_at": "2018-12-24T11:44:32.382Z", "updated_at": "2019-02-19T14:03:43.661Z", "last_modified_by_id": 202, "protocol_id": 3901 }, "step_comments": [ ], "assets": [ ], "step_orderable_elements": [ { "step_text": { "text": "\n\n

Make sure your target protein is dissolved in the liquid phase, and no inappropriate ingredients are present (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.


Mix samples with loading buffer. Mix by flicking the tube.

5X Sample buffer (Loading buffer):

\n\n






\n" }, "position": 0 } ] } ] } ], "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 } ] }