Binding Strength of Serum Proteins to Hyaluronic Acid Gels

• 1. Swell gels overnight in PBS to bring to equilibrium swelling.

  2. Incubate gels for 45 minutes at 37°C in 15% solution of FBS in PBS.

  3. Aspirate off FBS solution.

  4. Rinse three times with 500mL of DI-H₂O per gel. Allow each wash to sit on gels for 20 minutes at room temperature. Save supernatants from each rinse step in microcentrifuge tubes.

  5. Rinse once with 10% isopropanol. Allow isopropanol to sit on gels for 20 minutes. Save supernatants in microcentrifuge tubes.

  6. Rinse once with 30% isopropanol. Allow isopropanol to sit on gels for 20 minutes. Save supernatants in microcentrifuge tubes.

  7. Rinse once with 50% isopropanol. Allow isopropanol to sit on gels for 20 minutes. Save supernatants in microcentrifuge tubes.

  8. Rinse once with 70% isopropanol. Allow isopropanol to sit on gels for 20 minutes. Save supernatants in microcentrifuge tubes.

  9. Place open microcentrifuge tubes in hood overnight to allow isopropanol to evaporate.

  10. Freeze dry supernatants.

SDS-PAGE

• 1. Resuspend supernatants in 30mL SDS-PAGE sample buffer.

  2. Heat samples in boiling water bath for 4 minutes.

  3. Remove Ready Gel from storage pouch.

  4. Remove comb from gel and rinse with DI-H₂O.

  5. Cut along dotted line at the bottom of Ready Gel Cassette with a razor blade.

  6. Pull the clear tape at the bottom of the Ready Gel Cassette to expose the bottom edge of the gel.

  7. Repeat for second Ready Gel. (NOTE: use mini cell buffer dam if only one gel is to be run.)

  8. Place Gel Cassette Sandwich into electrode assembly with short plate facing inward.

  9. Slide electrode assembly into clamping frame.

  10. Press down on the electrode assembly while closing the 2 cam levers of the clamping frame.

  11. Lower the inner chamber into the mini tank. Fill the inner chamber with ~125mL of 1X SDS running buffer until the level reaches halfway between the tops of the taller and shorter glass plates of the gel cassettes. (NOTE: Do not overfill inner chamber.)

  12. Add ~200mL of 1X SDS running buffer to the Mini Tank.

  13. Load 10mL of sample into each well. Load samples slowly to allow them to settle evenly on the bottom of the wells. Remember to use special gel loading tips. Record what samples are in each lane. Always put MW marker in Lane 1 for orientation purposes. (NOTE: SIGMAMarker already contains sample buffer)

  14. Place lid on mini tank.

  15. Insert electrical leads into power supply.

  16. Turn on power and apply 150 volts. Run time is 25-35 minutes. Keep an eye on dye marker bands.

  17. When dye reaches the bottom of the gel, turn off power supply and disconnect the electrical leads.

  18. Remove tank lid and carefully lift out inner chamber assembly. Pour off running buffer. (NOTE: Running buffer can be re-used.)

  19. Remove gel cassette sandwiches from assembly.

  20. To remove the gel from a Ready gel cassette, slice the tape along the sides of the Ready Gel Cassette where the inner glass plate meets the outer plastic plate.

  21. The green plastic Gel Releaser may be used to pry the plates apart. Remove the gel from the glass plate by floating it off in fixative solution. Agitate gently until the gel separates from the plate.

  22. Stain the gel using Silver Stain Plus protocol. The fixative step is an acceptable stopping point if there is not enough time to finish the staining procedure.