小中大
Here is a protocol that I developed to purify small and hydrophobic peptides (4 kDa). The end purpose was for antibody production. Peptides were passively extracted from SDS gel slices. Cells (BL21 Star™(DE3) pLysS) were lysed at room temperature using 50 mM Tris-HCl (pH 8.0) and 5 mM EDTA. Inclusion bodies were collected at 12000g for 30 min at room temperature and followed by 25000g for 10 min after resuspending in the lysis buffer. The pellet was solubilized with Laemmli loading buffer and fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli 1970; Schägger 1994) using a Tris-Tricine buffer. Protein containing gel slices were frozen in liquid N2 and ground into small pieces. Expressed proteins were then eluted passively at 37°C by soaking in 1% SDS (~500 μl for every 200 μl Vol gel slice) for 1.5 h with constant motion. Eluted proteins along with SDS were injected into rabbits. Surprisingly, they were alive after injection and produced great antiserum. Hope this may help someone. Cheers,
