How to Use video for EasyPep Mini MS Sample Prep Kit

Add lysis buffer and universal nuclease to cultured cells or tissue samples. For cells, pipette up and down 10-15 cycles to reduce viscosity. For tissue, homogenize the sample and centrifuge at 16,000g for 10 minutes to collect the supernatant.

Determine protein concentration of the cell or tissue supernatant using a BCA protein assay. Transfer 10-100 micrograms of protein into a new microcentrifuge tube and adjust the final volume to 100 microliters with LC solution.

Add reduction solution and alkali solution to the sample, mixing gently between additions. Incubate at 95°C for 10 minutes using a heat block to reduce and alkalize the proteins.

Reconstitute Trypsin protease with enzyme reconstitution solution and add 50 microliters to the reduced/alkalized sample. Incubate at 37°C for 1-3 hours with shaking to digest the proteins, then add digestion stop solution.

Remove the white plug from the bottom of the peptide cleanup column, place in a microcentrifuge tube, and centrifuge at 300g for 2 minutes to remove all liquid from the column.

Transfer the protein digest (approximately 300 microliters) into the conditioned peptide cleanup column and centrifuge at 1500g for 2 minutes. Wash the column twice with wash solutions A and B, centrifuging at 1500g for 2 minutes each time and discarding flow-through.

Transfer the peptide cleanup column to a new microcentrifuge tube and elute with elution solution by centrifuging at 1500g for 2 minutes. Dry the peptide sample using a vacuum centrifuge, resuspend in 0.1% formic acid, and quantify peptide concentration before LC-MS analysis.