Hb Buffer:

  • 50 mM Tris, (pH 7.0 with HCl @ 21° C)
    • Volume of stock needed for final solution = 75 mL
  • 40 mM NaCl
    • Volume of stock needed for final solution =0.120 mL
  • 10 mM Mg2+
    • Volume of stock needed for final solution =0.15 mL

Sample Preparation (@ ~1° C):

  • Tube 1:
    • Rinse worm in dh water
    • Place worms in ice in order to reduce movement
    • Sever head @ segment 6
    • Pipette 5 µL blood
    • Mix blood in 40 µL Hb buffer
    • Wait 30 minutes – 1 hour to find/setup equipment
    • Spend 30 minutes freezing grids
  • Tube 2:
    • Place worms in ice in order to reduce movement
    • Rinse worm in dh water
    • Sever head @ segment 6 (see below)
    • Pipette 5 µL blood
    • Mix blood in 500 µL Hb buffer
    • Wait 30 minutes – 1 hour to find/setup equipment
    • Spend 30 minutes freezing grids

Cryo Plunging:

  • 4 µL sample for 1-2 minutes
  • Q-foil (2/2 – 400 mesh)
  • Glow discharged (1 minute, 13 w, Solarus)

Lumbricus Terrestris Anatomy

earthworm internal anatomy Fresh Earthworm Diagram

Hemoglobin Single Particles and Structure Example

Chen, W.T., Chen, Y.C., Liou, H.H., and Chao, C.Y. Structural basis for cooperative oxygen binding and bracelet-assisted assembly of Lumbricus terrestris hemoglobin. Scientific Reports, 2015, 5:9494 (doi: 10