Silver stain 2 kit wako

The fat body collected from 10 silkworm larvae was suspended in 25 ml of ice-cold TBS buffer (pH 7.4) and lysed by sonication 3 times for 30 s, with 1-min intervals between each. The sonication condition was 40 amplitudes with 4 output watts (Sonics and materials Inc., CT, USA). The sample was then centrifuged at 20,000 × g for 20 min, and the supernatant was filtered using a 0.45 µm filter. The collected filtrate was used for affinity purification using DDDDK tagged protein purification gel (Medical and Biological Laboratories Co., LTD, Nagoya, Japan). The DDDDK tagged protein purification gel was equilibrated with TBS buffer prior to use. The collected supernatant was mixed with 1 ml of gel and gently stirred at 4°C for 1 h. This mixture was centrifuged at 2500 × g for 5 min, and the precipitated resin was washed with 36 ml of TBS buffer. The proteins bound to the resin were eluted with elution buffer (0.1 M glycine, pH 3.5). To check the expression of Polh-Cit1a fusion protein, 50 µg of purified fusion protein sample was digested with 1 unit of recombinant enterokinase (rEK; Novagen, Darmstadt, Germany) at room temperature for different time intervals. The product was analyzed by SDS-PAGE and silver staining according to the company's protocol (Silver Stain II Kit Wako, Wako Pure Chemical Industries, Ltd. Tokyo, Japan).