Table 2.

Troubleshooting Guide for Proteome Profiling of Neutrophils

Problem	Possible cause	Solution
Low neutrophil enrichment yields	Inefficient negative selection	Add an additional magnet capture step to the purification protocol
Low viability of PMNs <75%	Extended purification time or Erythrocyte lysis	Avoid red cell lysis
Poor digestion of peptides	Concentration of Urea is too high during trypsin digestion	Ensure that urea is at 2 to 2.5 M during trypsin digestion; check pH during digestion (pH 8 is optimal)
	Lys-C and trypsin proteases are not working optimally	Use fresh proteases stored according to manufacture’s instructions. Keep proteases on ice at all times during use and discard any remaining protease after thawing.
Poor peptide yield	In-house STAGE-tips were packed too tightly/loosely	Ensure recommended centrifugation speeds and times are accurate for Buffer A; if tip is packed too loosely, Buffer A will pass very quickly and if packed too tightly, higher speeds and time will be required to wash the tip
	STAGE-tip is not sufficiently activated or has run dry during loading	Ensure that C18 resin never runs dry during activation, loading, or washing