Sample prep and quality assurance

Harms Lab Wiki > Protein Biochemistry > > Sample prep and quality assurance

Preparing proteins for experiments

  1. Thaw protein pellets.
  2. Centrifuge @13,000 rpm for 10 minutes @ 4 C in the refrigerated centrifuge.
  3. If necessary, concentrate your protein using a Pall centrifuge filter.  (Make sure you choose the right molecular weight cutoff so you do not lose your sample!).
  4. Buffer exchange using NAP-25 columns.
  5. Equilibrate two columns, each with 25 mL of your buffer.
  6. Load 2.5 mL of sample onto the first column.  Let it entirely enter the column bed.
  7. Add 1 mL of buffer as void volume.
  8. Elute sample with 2.5 mL more buffer.
  9. Repeat steps 5-7 on the second NAP-25 column.
  10. Measure the absorbance of your final protein and verify that there is no scatter.

Quality check

  • Is the buffer right?  If you’re dealing with divalent metals, was the buffer treated with chelex and filtered at 0.22 um?
  • Measure the protein concentration and verify that there is no scattering signal at 320 and 340 nm.
  • Check that protein purity is >99% by SDS-PAGE.
  • Verify that the protein is in the apo form using intrinsic Tyr fluorescence.  (Measure the emission spectrum of your protein.  Add 2 mM EDTA and remeasure.  These spectra should be identical.  Add 10 mM CaCl2.  For most S100s, this should give a large increase in fluorescence signal).
  • Do you need to add reducing agent?  (Be careful if you’re using a transition metal, as these will be preferentially reduced!)
  • Was the clone from which this protein was prepped ever sequenced?
  • Was the identity of the purified protein verified by mass spectrometry?
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