Page 1 of 2 Experiment 5 –
SDS-PAGE & Western Blotting of Marine Proteins SDS-PAGE AND TRANSFER –
Week Two WESTERN BLOT (or Immunodetection) 1. Take the plastic bag with your blot and place the entire thing in a tray. Add 25 mL of blocking solution and seal the bag. (If a little blocking solution leaks out into the tray, that
’s fine.)
. Place the tray on a rocker set at a low but steady speed (about 3 or 4) and incubate for 10 min at room temperature. 2. Pour off the blocking solution from the bag and
any solution that leaked into the tray. 3. Add 10 ml of the anti-myosin 1° antibody solution to the plastic bag with the blot and seal the bag. Place the tray with the bag and blot back on the rocker and incubate for 10 min at room temperature. Make sure the 1° antibody does not leak from the bag! 4. Pour off the 1° antibody solution. Add 50 ml of wash buffer to the plastic bag with the blot, seal and agitate the tray to rinse. Pour off the wash buffer. 5. Add another 50 ml of wash buffer to the bag with the blot, place in the try, and place on the rocker for 5 –
10 min. (If a little wash buffer leaks out into the tray, that
’s fine.)
6. Pour off the wash buffer from the bag and any that leaked into the tray. And add 10 ml of goat-anti-mouse-HRP 2° antibody solution to the bag with the blot. Place back in the tray and put back on the rocker. Adjust the rocker to a faster setting and incubate for 10 min at room temperature. Make sure the 2° antibody does not leak from the bag! 7. Pour off the 2° antibody solution from the bag. Rinse the membrane by agitation in 50 ml of wash buffer. Pour off the wash buffer. 8. Add another 50 ml of wash buffer to the bag and place on the rocker at a slower speed again. Incubate for 5 –
10 min. (If a little wash buffer leaks out into the tray, that
’s fine.)
9. Pour off the wash buffer from the bag, and any buffer that leaked into the tray. 10. Add 10 ml of HRP colour detection reagent to the bag with the blot and place in the tray. Place the tray on the rocker. Incubate for at least 10 min to allow for coloured bands (pink to violet) to develop. If there is time, wait for 30 min for optimal colour development. 11. Once the bands have developed, discard the detection reagent and rinse the membrane twice with distilled water. 12. Image the blots using the Gel Doc imaging system and the white sample tray
. Make sure to save the image onto a flash drive and print a copy. You will need this as part of your ‘Observations –
Week 2
’.
