In brief, the sample was grinded in lysis buffer for 3 min, centrifuged at 700 g for 5 min, resuspended in lysis buffer, precipitated in medium buffer, and stored in store buffer.
Reaction buffer B was used in the T7 DNAP-unwinding assays as SSB was observed to precipitate in buffer A. The final concentrations of enzymes and DNA were 10 nM fork DNA, 20 nM T7 helicase hexamer, 20 nM T7 DNAP, 200 nM E. coli SSB, and 2 μM dT90 trap DNA.
The procedure that we arrived at consists in simply adding one volume of a calibrated precipitation buffer, followed by centrifugation to pellet precipitates, and exchanging the buffer (Scheme S1).
For this particular formula to precipitate, the buffered chromate solution needs to be around pH 6 (achieved by the addition of the potassium carbonate) when the zinc nitrate is added.
After washing the beads with lysis buffer, precipitated proteins were resolved in sample buffer, boiled, separated by 4%, 3 8% or 4 12% gradient NuPAGE pre-cast gels (Invitrogen), transferred onto PVDF membranes, and analysed with the indicated primary and secondary Abs.
After four times washes with the lysis buffer, precipitated proteins were eluted in lysis buffer containing 6 M urea.
The crystallization conditions for Pfu and Ssol SRP54s are quite similar (high concentration of lithium sulfate as precipitating agent in acetate buffer at an acidic pH but no detergent in our case).
Since Mn2+ precipitates easily in buffer pH > 10, we chose pH 9.0 which gave the largest peak amplitude and best reproducibility.
Briefly, proteins were precipitated from lysis buffer with nine volumes of methanol, pelleted by centrifugation, washed with 1 ml of 90% methanol, dried and dissolved in sodium dodecyl sulfate sample buffer at the final concentration of 0.1 μg protein/μl.
