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Lab. Bioclock & Aging/Dewan Md. Sumsuzzman Bradford assay ⎸Protein quantification A. Preparation of BSA standard curve Protocol: Preparation of 10 mg/ml BSA solution (10 ml DW + 100 mg BSA) [ Stock solution ] Prepare 1 mg/ml BSA solution (9 ml DW + 1 ml of 10 mg/ml BSA) [ Working solution ] Levelling total 8 cuvettes as Blank, 1, 2, 4, 6, 8, 10, 12 µL respectively. For blank, add 1 mL lysis buffer. And, add 1 mL working solution to 1, 2, 4, 6, 8, 10, 12 µL levelled cuvettes respectively. Discard 1, 2, 4, 6, 8, 10, 12 µL working solution from 1, 2, 4, 6, 8, 10, 12 µL levelled cuvettes gradually. Start quantification and get a BSA curve. B. Protein quantification Protocol: Part-1 (Prepare working solution): Prepare lysis buffer (Western blot protocol) Mix the tissue with lysis buffer in a conical tube (10 mg tissue/1 mL lysis buffer) and incubate for one hour. Prepare bradford working solution by mixing bradford stock solution and autoclaved DW with a ratio of 1:4 [{Dilution formul

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman  ECL development Protocol Remove ECL Western Blotting Reagent Kit (Pierce® ECL plus western blotting substrate) from the refrigerator. Prepare the substrate working solution by mixing Substrate A and Substrate B in a 40:1 ratio (example: 12 mL substrate A + 300 µL substrate B for 8 × 12 cm2 membrane). Use 0.125mL working solution per cm2 of membrane. Note: The working solution is stable for up to 1 hour at RT. Place the membrane on the other suitable clean surface, remove the water, add Pierce® ECL plus western blotting substrate (working solution) and spread evenly over the membrane surface. Incubate blot with working solution for 5 minutes at RT. Remove blot from working solution and place it in a plastic sheet protector or clear plastic wrap. Use an absorbent tissue to remove excess liquid and carefully press out any bubbles from between the blot and the membrane protector. Place the protected membrane in a film cassette with the prot

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman Antibody incubation & Washing ※ When you first take the Antibody, it tells you how much concentration should be diluted. Use it at that concentration. If the protein band comes out, continue to use it. If the band does not come out, increase the concentration. Primary & secondary antibody incubation ※ If there are several proteins you want to see, the membrane may be cut to size. Prior to Primary Ab treatment, remove the water, wrap it in a wrap, and check for protein markers (total volume 2-3 ml). A. Dilution of primary Ab: Dilute primary Ab in TBST (0.5% Skim milk / TBST) and incubate shaking incubation for 1 hour at room temperature. Or overnight at 4 ° C. → 0.5% skim milk + TBST + primary Ab → Used primary Ab can be re-used, can be kept at 4 ℃ Ex 1) TBST 10 ml + Skim milk 0.05 (g) + 10 μl of primary AB when the dilution concentration is 1: 1000 and the total volume is 10 ml Ex 2) TBST 2 ml + Skim milk 0.01 (g) + 2 ㎕ of primar

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman Transfer membrane Preparation: • 10X & 1X Transfer buffer (4℃) • Sponge / Filter paper / 3MM paper / Membrane • Cassette / Blotting chamber • 10X TBS (pH 7.5) &  1X TBST • 0.2% Ponceau S • Coomassie blue • Blocking buffer (5% skim milk in TBST) • Staining solution & Destaining solution • BM purple AP substrate Transfer protocol: Cut the membranes to the appropriate size and immerse them in transfer buffer. For PVDF membrane, immerse in methanol for 5 minutes before transferring to transfer buffer. Membrane activation:  The process by which proteins in the gel make hydrophobic interaction with the membrane. This process is not necessary when using an NC. Transfer the electrophoresis gel to the transfer buffer, remove the wells, and remove the remaining portion except the sample. → Save membrane. Place the black portion of the Western blot cassette against the bottom of the container containing the transfer buffer and place it

Lab. of Bioclock & Aging/Dewan Md. Sumsuzzman Ponceau S Staining Ponceau S Staining Solution: Table-1: Recipe of 0.1%  (w/v) Ponceau S in 5% (v/v) Acetic acid Ingredients Quantity Ponceau S 1 gm Acetic acid 50 ml ddH2O Make up to 1L Table-2: Recipe of 0.2%  (w/v) Ponceau S in 3% (v/v) Acetic acid Ingredients Quantity Ponceau S 2 gm Acetic acid 30 ml ddH2O Make up to 1L Table-3: Recipe of 0.1N NaOH in 100 ml water Ingredients Quantity NaOH 0.4 gm H2O 100 ml Protocol: Following Western blotting, transfer the membrane to 5ml Ponceau S Stain solution. Place on an orbital shaker for 5 minutes at room temperature. Rinse membrane with DI water to achieve desired staining, approximately 2‐3 washes of 5 minutes each will remove the background staining. For total protein destaining, use a 0.1N NaOH solution. Wash the membrane with 0.1N NaOH solution for 5 minutes. Protein bands will start disappear after 10-30 seconds. Discard wash solution and repeat once. Wash the membrane 2‐3 times with

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman Coomassie Blue Staining Table-1: Recipe of Staining Solution Ingredients Quantity Coomassie Blue R-250 2.5 gm Methanol 500 ml Acetic acid 100 ml DW 400 ml Note:  Final volume 1L. Add 100 ml of acetic acid to 500 ml of ddH2O. Add 500 ml of methanol and mix. Add 1g of Coomassie R250 dye and mix.  Put a magnetic bar in a brown bottle, and add dye while spinning. Finally, filter it. Store at room temperature in a sealable container. Table-2: Recipe of Destaining Solution Ingredients Quantity Methanol 500 ml Acetic acid 100 ml DW 400 ml Store at room temperature in a sealable container. Protocol After the gel has finished running, remove it from the tank and remove the gel from the glass plates. Wash the gel 2-3 times, 5 minutes each in deionized water to remove SDS present in the gel. Each wash should be large volumes of water. Remove all free water from the gel. Add an adequate amount of staining solution (Coomassie Blue R-250) to cover the

Lab. Bioclock & Aging/Dewan Md. Sumsuzzman Gel Electrophoresis Preparation of Gel: Part-1: Clean glass plates with D.W and 70% ETOH, dry thoroughly and assemble them. Place 100% ETOH between glass plates to check for leakage. If the ETOH does not leak, clean the ETOH using 3MM paper. Carefully insert the comb, mark it in the comb down to 0.5 cm, and remove the comb. Part-2: Identify the molecular weight of the protein to be separated and determine the concentration of acrylamide. Table-1: Acrylamide concentration as per protein size. Acrylamide concentration (%) Protein size (kDa) 15 10-43 12 12-60 10 20-80 7.5 36-94 5 57-212 Table-2: Preparation of running gel. Running Gel (10 ml) 5％ 7.5％ 10％ 12％ 15％ D.W H2O 5.3 mL 4.6 mL 4 mL 3.3 mL 2.3 mL 1.5 M Tris-Cl (pH 8.8) 2.5 mL 2.5 mL 2.5 mL 2.5 mL 2.5 mL 30 acrylamide Solution 2 mL 2.7 mL 3.3 mL 4 mL 5 mL 10 SDS 100 µL 100 µL 100 µL 100 µL 100 µL TEMED 8 µL 6 µL 4 µL 4 µL 4 µL 10 APS 100 µL 100 µL 100 µL 100 µL 100 µL Note:  Before add