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EMBL Hamburg Biological
Small Angle Scattering
BioSAXS

Refractometer measurements

How to make a protein concentration measurement with the Anton Paar Abbemat 550 refractometer

Note this procedure describes the estimation of protein concentration using RI of samples in aqueous (water) buffer. Sample volumes as low as 4 μl can be measured and recovered. Never attempt to measure samples in organic solvents and never clean the prism with an organic solvent.

  • Switch on the instrument. The on-switch is located at the back of the instrument on the left hand side.
  • Wait at least 15 minutes to let the system boot up and for the optics and sample state to equilibrate to 20°C. During this time, remove the black cap covering the prism (sample stage) and thoroughly wash the stage with water. Dry the prism using a fine tissue that does not leave behind dust (e.g., a Kimwipe).
  • Make sure the prism is completely dry.
  • Place 4-5 μl of buffer onto the centre of the prism without introducing any bubbles. Use a PLASTIC pipette tip. Never use a needle!
  • Press 'Measure' on the front panel. Wait 15 seconds or until the instrument beeps and the measurement is classed as valid.
  • Record the number on the front to all decimal places - this is the refractive index of the buffer (nD).
  • Clean and dry the prism using water and a tissue.
  • Place 4-5 μl of protein sample onto the centre of the prism without introducing any bubbles.
  • Press 'Measure' on the front panel. Wait 15 seconds or until the instrument beeps and the measurement is classed as valid.
  • Record the number to all decimal places - this is the refractive index of the protein sample (nD).
  • Clean and dry the prism once again.
  • Subtract the buffer nD from the sample nD.
  • Divide the resulting number by 0.19.
  • The result is the protein concentration in g/ml.

When you have finished measuring:

  • Replace the protective black prism cap.
  • Switch off the instrument.

  Last modified: October 4, 2017

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