Preparing proteins for experiments
- Thaw protein pellets.
- Centrifuge @13,000 rpm for 10 minutes @ 4 C in the refrigerated centrifuge.
- 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!).
- Buffer exchange using NAP-25 columns.
- Equilibrate two columns, each with 25 mL of your buffer.
- Load 2.5 mL of sample onto the first column. Let it entirely enter the column bed.
- Add 1 mL of buffer as void volume.
- Elute sample with 2.5 mL more buffer.
- Repeat steps 5-7 on the second NAP-25 column.
- 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?
Category: Protein Biochemistry