Blocking buffer:
\nSoak 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:
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:
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.
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.
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.
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.
Remove the gel assembly from the electrophoresis apparatus. Remove the gel from the glass plates using a spatula, and prepare for subsequent analysis.
", "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": "\nSet 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.
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": 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" } ] }, { "step": { "id": 5250, "name": "Make the stacking gel", "description": "\n\nProteins 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:
Heat the samples at 100°C for 3 minutes in a heat block.
", "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": [] }, { "step": { "id": 5251, "name": "Mix samples with buffer", "description": "\n\nMake 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):