Individual DNA samples were pooled (50 ng per individual) and subsequently concentrated using a DNA concentrator Eppendorf 5301.
Refolding was attempted using the detergent dilution method that has been successful for refolding other helical membrane proteins [20,21]; the SDS concentration was diluted to below its cmc with 20 mM Tris buffer, pH 7.5 containing 0.1% DDM and subsequently concentrating the sample using a concentrator with a 100 kDa cut-off membrane (Millipore) to ∼ 100 μl.
The fractions containing proteins (MBP-ΔN597 or MBP-ΔNCP69) were confirmed by SDS PAGE, and were subsequently concentrated using 10 kDa MWCO Vivaspin Sample Concentrators (Sartorius Stedim Biotech).
PolyA RNA was subsequently concentrated by ammonium acetate ethanol precipitation and concentrations were determined using the Qubit fluorometer (Invitrogen, Carlsbad, CA) and the Quant-iT RNA Assay Kit (Invitrogen, cat#Q33140).
Purified DBLs were subsequently concentrated by means of Macro- and Micro-sep concentrators.
The eluted and neutralized samples were combined, diluted to 20ml with MilliQ water and subsequently concentrated to the original starting volume using centrifugal concentration tubes [Vivaspin 20, Sartorius AG, Goettingen, Germany: Code no VS2021].
The protein was cleared of imidazole using dialysis with phosphate buffered saline and subsequently concentrated using a 9 kDa cut-off protein concentrator according to the manufacturer's instructions (Thermo Fisher Scientific, Rockford, IL, USA).
Mr. Kluge, 87, subsequently concentrated on filmmaking, restaurant and telecommunications ventures.
The collected media were subsequently concentrated, using a cut-off filter of 3 kDa.
Subsequently concentrating on the three-cushion game, he won 12 annual world championship tournaments (1936, 1940 44, 1947 52).
