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iGEM Sumbawagen 2014 · Econey

Notebook – Protocol – 13. SDS-PAGE

    1. 1 ml of E. coli BL21(DE3) overnight culture was added to 1.5 ml propylene tube. Then centrifuge at 6,000 rpm, 5 minutes at room temperature. Then supernatant was discarded.

    2. Pellet was added with 300 ul of 1x Bug Buster (Novagen). Tube was vortexed.

    3. Tube was put on boiled water for 5 minutes.

    4. 50 ul of samples was aliquoted and used as “total protein” sample.

    5. The rest of the sample in the tube was centrifuged at 6,000 rpm, 10 min, room temperature.

    6. 50 ul of supernatant was aliquoted and used as “soluble protein’ sample.

    7. Protein concentration of “total protein” and “soluble protein” were estimated using Lowry method based on the kit from Biorad. We have no UV spectrophotometer for measurement of real concentration at 750 nm. But according to our chief instructor Dr. Arief previous experiment, the concentration of “soluble protein” will be about 1,000 – 2,000 ug/ml, while the concentration of “total protein” will be about 10,000 – 20,000 ug/ml.

    8. “Soluble protein” sample was used directly, while “total protein” sample was diluted 1/10th to obtain samples with roughly the same concentration for SDS-PAGE.

    9. 15 ul sample was added with 15 ul dye containing SDS and Coomassie Brilliant Blue (CBB) in 500 ul polypropylene tube, then the tube was put in boiled water for 15 min

    10. 15 ul of sample – protein quantity about 10 ug – was electrophoresed. Marker used was Blue Star Prestained Protein Marker (Nippon Genetics). The marker has proteins bands of 170 / 125 / 93 / 68 / 53 / 41 / 32 / 23 / 14 / 10 kD.

    11. SDSPAGE was done with commercial 10-20% SDS gel, and 1x SDS buffer. 10x SDS buffer was prepared using 250 mM Tris, 1,920 mM Glycine, and 1% w/v SDS.

    12. SDSPAGE was done for 45 minutes with equipment setting of 40 mA.

    13. Staining of SDSPAGE gel was done using Rapid CBB kit (Kanto Chemicals). Staining was done 1-3 hours.

    14. Destaining of SDSPAGE gel was done using solution containing 75 ml distilled water, 10 ml acetic acid, 15 ml ethanol. Destaining was done overnight.

    15. Gel was documented with pocket camera, and file was evaluated with Photoshop program in a computer.