Omni bead ruptor 24 elite

To examine our newly developed direct method, we compared it to a previously published NaOH‐based SDS repeated extraction method 8. We modified the published protocol to fit the purpose of protein extraction from a single hair shaft. The modified workflow was performed as follows (also illustrated in Appendix S1): (i) First, we used bead milling for sample preparation instead of incubation with lysis buffer: 5‐cm‐long hair shafts are ground by a bead mill (Omni Bead Ruptor 24 Elite; Omni International Inc., Kennesaw, GA) repeatedly (3 cycles, 30‐second grinding at the speed of 5 m/s and 30‐second dwell); (ii) next, ground hair samples are incubated with a NaOH‐based lysis buffer that contains SDS and beta‐mercaptoethanol (ßME) for three cycles according to published 8 protocol, and in each cycle, the hair residue is recycled through the process with bead milling; (iii) pooled supernatant containing hair proteins are precipitated with acetone; (iv) pellets from protein precipitation and leftover hair debris are combined for downstream SDS‐PAGE; and (v) in‐gel digestion was used to generate peptides.

From each granularized sample, we transferred a 10 mg aliquot to a 2 mL beadmill tube (BioSpec, Bartlesville, OK) and added 1 mL of lysis buffer (buffer AP1; QIAGEN, Venlo, Netherlands) and ~0.5 mL of chrome steel beads (1.3 mm diameter; BioSpec, Bartlesville, OK). We then processed the samples on an Omni Bead Ruptor 24 Elite beadmill (Omni International, Kennesaw, GA) using a speed of 6 m/s and repeating for four 1-min runs, cooling samples on ice between runs. The resulting lysate was then purified using a Qiagen DNeasy Minikit according to the manufacturer’s protocol, and DNA yield was verified on a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). Importantly, our choices of bead size, bead material, and beadmill run settings were based on preliminary trials in which we observed that beadmill homogenization with other combinations of parameters did not necessary lyse all grains and often lysed grains in a taxon-biased manner.