Table 16.

Washing Twice with Buffer Containing 50 mM ABC and 8 M Urea

Processing time	Step	Comments / Tips
1–2 hours	Add 200 μL 50 mM ABC + 8 M urea to each sample filtration unit. Centrifuge units for 15 minutes at 12,000 × g.	There are many steps requiring centrifugation. It is helpful to check off which steps have already been completed to keep track. Keeping an eye on the level of eluted solution can also be helpful since waste should be emptied every two spin cycles (so waste should be emptied a total of 2 times before switching to final collection tubes.). Sometimes, due to filter clogging, the filtration rate may be rather slow. In such a situation, it may be ok to increase the centrifugation speed (for any of the steps requiring centrifugation) to 12,500 × g or 13,000 × g, but no more. A number of washing steps are encountered in this workflow. Based on the buffer being used, these steps help to clean and denature the protein, getting the sample ready for proteolytic digestion. Note that some buffer components, like urea, are great for protein denaturation but must be removed from the samples prior to proteolytic digestion to prevent inhibition of the protease.
	Empty eluent into waste so that it does not reach a volume that touches the filter. Handle the filter very carefully.
	Repeat the wash with another 200 μL 50 mM ABC + 8 M urea buffer. Centrifuge units for 15 minutes at 12,000 × g.