Nah2po4

An equal number of cells was lysed in 1 ml of guanidinium lysis buffer
(6M guanidinium-HCl, 0.1M Na₂HPO4 (Sigma)/NaH₂PO4 (Fisher)
pH 8.0, 0.01 M Tris pH 8.0, 5mM imidazole, 10mM β-mercaptoethanol
(Fisher)). Lysates were incubated with 45 μl of Ni2+-NTA-agarose
beads (Thermo Scientific) for 4 hrs at room temperature by end-over-end
rotation. The beads were washed with the following buffers: guanidinium buffer
(6M guanidinium-HCl, 0.1 M Na₂HPO4/NaH₂PO4 pH 8.0, 0.01 M
Tris pH 8.0, 10 mM β-mercaptoethanol); urea pH 8 buffer (8M urea
(Fisher), 0.1 M Na₂HPO4/NaH₂PO4 pH 8.0, 0.01 M Tris pH
8.0, 10 mM β-mercaptoethanol); buffer A (8M urea, 0.1 M
Na₂HPO4/NaH₂PO4 pH 6.3, 0.01 M Tris pH 6.3, 10 mM
β-mercaptoethanol) plus 0.2% Triton-X 100 (Acros); buffer A;
buffer A plus 0.1% Triton X 100. Proteins were eluted with 200 mM
imidazole in 5% SDS (Fisher), 0.15 M Tris pH 6.7, 30% glycerol
(Fisher), 0.72M β-mercaptoethanol. Samples were then subjected to
SDS-PAGE and Western blotting.

Dithiothreitol (DTT), iodoacetamide (IAA), formic acid (LC-MS grade), Na₂HPO₄, and NaH₂PO₄ were purchased from Thermo Fisher Scientific (Waltham, MA). Ammonium bicarbonate (ABC) buffer (50 mM) was freshly prepared from a 500 mM stock solution. LC-MS grade water and acetonitrile were purchased from Honeywell (Charlotte, NC). Trypsin gold and rLys-C (MS grade) were from Promega (Madison, WI). Scott’s tap water substitute bluing reagent was from Ricca Chemical (Arlington, TX). Ethanol was purchased from Decon Laboratories, King of Prussia, PA, and paraffin wax was from Blended Waxes, Inc. (Oshkosh, WI). Other chemicals were from Sigma-Aldrich (St. Louis, MO). Neutral buffered formalin (pH 6.8–7.0) was prepared by combining 25 mL (37–40% w/w) of formaldehyde with 225 mL of purified water, 1.0 g of NaH₂PO₄, and 1.625 g of anhydrous Na₂HPO₄. Carboxymethylcellulose solution (CMC; 2.5% w/v in water) was prepared for embedding fresh samples prior to freezing. A stock solution of n-dodecyl-β-d-maltoside (DDM, Sigma-Aldrich), a nonionic surfactant used for protein extraction and solubilization,^{36 (link)} was prepared at 1% (w/v) in water and further diluted for use, as described below. Nanowell chips were fabricated as described previously.^{17 (link)}