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Calmodulin Purification Protocol

(By Dilini Kurukulaarachchi, adapted from Matthew Sigurdsons 2010-2011 lab notebook, originally from Dr. Qilu Ye)

Buffers required: Buffer Components A 25 mM Tris, 1 mM EDTA, 1 mM PMSF*, 1 mM DTT B 25 mM Tris, 1 mM CaCl2 C 25 mM Tris, 2 mM CaCl2, 200 mM NaCl D 25 mM Tris, 1 mM EGTA * PMSF will not dissolve in water, so must dissolve in 95% EtOH pH (with HCl) 7.5 7.5 7.5 7.5

1. Cell Resuspension & Lysis: Resuspend pelleted cells in Buffer A (300 mL/L culture) NB: BEFORE adding lysozyme, note that it will be difficult to distinguish CaM and lysozyme on gel (even 16%), so if testing for the presence of CaM, dont add lysosyme (also applies for proteins that are co-purified with CaM) since CaM MW is ~14-17 kDa** and lysozyme is ~15 kDa **CaM changes apparent MW depending on (+)/(-) Ca2+ (seen on Matthews gels): +Ca2+: 14-15 kDa +EDTA: 16-17 kDa Add 5mL lysozyme / 1 L culture (read note above in advance) Add 50 uL DNAase I / 1 L culture Incubate 30 mins with shaking Store in -80C or move to next step 2. Lysis: Freeze-thaw 3x If pellet already frozen, first round thawing counts as 1 cycle Materials required: liquid nitrogen, bucket, metal holder for manoeuvring conical once in dry ice (wear gloves!); warm water bath Procedure: take pellet and move into bucket filled with liquid nitrogen until crackling stops (freezing complete); move into warm waterbath (to thaw); repeat Sonicate (1 min, 30%, 5 sec pulse on/off) Centrifuge: 16,000 rpm, 30 mins 3. Crude Purification by Boiling: Materials required: Bunsen burner, lighter/flint, tripod, 1 L glass beaker, thermometer, boiling stones (optional) Add water to glass beaker and preheat on Bunsen burner (with beaker sitting on top of tripod), add few boiling stones and monitor temperature with thermometer (dont go over 90C) Decant supernatant (S1) into another conical

2 of 2 Collect supernatant (S1) and pellet (P1) samples for SDS-PAGE (optional) Add 100uL of 1 M CaCl2 per 50 mL supernatant (key step! Otherwise sample wouldnt precipitate) Place conical containing S1 into boilng water and ensure heating 85-90C; this should form lots of white precipitate When no more precipitation forming, centrifuge: 16,000 rpm, 20 mins Can wash column in step 4 during this time Syringe filter supernatant (S2) into different conical to remove precipitate that is still resuspended Collect supernatant (S2) and pellet (P2) samples for SDS-PAGE (standard) 5. Hydrophobic chromatography (using phenyl-Sepharose column) [use peristaltic pump to achieve faster, but ensure no column drying!] Pre-wash phenyl-Sepharose column with Buffer B Load supernatant (S2) & collect flow through (FT) Wash: W1: 3x with Buffer B W2: 2x with Buffer C W3: 3x with Buffer B Elute (E) CaM with Buffer D Collect FT, W1, W2, W3, E samples for SDS-PAGE 5. Run SDS-PAGE gel (16%) to confirm purification was successful: Total samples 1 ladder + 7-9 purification samples = 8-10 wells required (Gel filtration could be used after dialysis for further purification; or if studying CaM interaction with another protein, could do gel filtration together with that protein) 6. Concentrate E to ~1 mL (no higher) 7. Determine concentration of CaM: -UV: = 2980 M-1 cm-1, at 280 nm measured in water; try 100x dilution if concentrated to 1 mL -measuring by UV only gives 64% of protein detected by Bradford; ProtoParam warns that computed likely contains >10% error -Bradford: try dilutions 100x and 200x if concentrated to 1 mL 8. Flash-freezing: Pick up cryo-vial(s) and label: CaM, date, concentration, your name; place bottom on liquid nitrogen ensuring none gets into vial 30 uL aliquots flash-frozen with liquid nitrogen Store at -80C