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How to Prepare a Hanging Drop

To determine the 3D structure of a protein, you first need a pure crystal of it. The hanging-drop method uses a drop of protein-containing solution hanging from a coverslip. Beneath this hanging drop lies a reservoir with a higher salt concentration than that of the drop. Over time, water vapor leaves the drop and ends up in the reservoir, causing the protein to crystallize. Each protein requires different conditions (such as salt concentration and pH), and often trial and error offers the only way to find the right conditions.

Things You'll Need

Lab coat, gloves and eye protection
Micropipette with pipette tips
1 molar solution of sodium chloride in 0.05 molar acetate buffer, pH 4.7
2 mL Eppendorf tubes (or similar)
Centrifuge
24-well plate
Vacuum grease in syringe
Siliconized coverslip
Compressed air
Tweezers
Clay (e.g. modeling clay or similar)
50 mg / mL lysozyme in 0.05 molar acetate buffer solution, pH 4.7

Instructions

- 1
  Put on lab gear (coat, gloves and eye protection).
- 2
  Use a micropipette to add 1.1 mL of a sodium chloride (salt) and acetate buffer solution into an Eppendorf tube.
- 3
  Centrifuge the closed Eppendorf tube with the salt solution inside it for about five minutes at 13,000 rpm. Remember to balance the centrifuge by placing another Eppendorf tube containing 1.1 mL of water across from and opposite to the salt solution tube.
- 4
  Add 1 mL of the salt solution to a well in a 24-well plate.
- 5
  Use a grease-filled syringe to place a thin layer of grease around the rim of this well. Take care not to add too little or too much. If you add too much grease, the grease will run down into the well and possibly ruin your experiment. If you use too little, the well will not be airtight and your experiment will not work. Aim for just enough grease that the coverslip and grease form an airtight seal later on.
- 6
  Roll the clay with your hands to make four small balls, and push them onto the four corners of the 24-well plate. They will act as supports to ensure the cover of the plate doesn't come into contact with the coverslip.
- 7
  Use compressed air to blow any dust off of the coverslip. Hold the coverslip with a pair of clean tweezers.
- 8
  Suck up 4 microliters of the lysozyme solution with your micropipette, and add this solution to the center of the coverslip. Change pipette tips, and add 4 microliters of the salt solution. Gently pipette up and down, sucking in fluid then expelling it, to mix the salt solution with the lysozyme solution. Take care to mix the two without dispersing the fluid all over the coverslip.
- 9
  Turn the coverslip over quickly so the fluid doesn't slide off, and plant it squarely atop the well. Take care to press gently on it so it forms an airtight seal with the grease all around the edges of the well.
- 10
  Push the cover of the 24-well plate onto the clay balls, and leave the plate in a cool place.

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