Pro q diamond phosphoprotein enrichment kit

Phosphorylated proteins were enriched from cell lysates with a Pro-Q Diamond Phosphoprotein Enrichment Kit (Invitrogen) according to the manufacturer's protocol. Briefly, cells were lysed in lysis buffer supplemented with inhibitors of endonuclease and proteinase. Using non-denaturing conditions, lysates were loaded on the columns, concentrated and finally precipitated with methanol and chloroform. The resulting pellet was dried and resolubilized with SDS sample buffer. Tec or phosphorylated Tec, as well as a loading control were detected by immunoblot analysis.

In the first approach, the procedure described by Pink and coworkers [6 (link)] was adapted to enrich the phosphoproteome of 4 randomly chosen PBMCs samples. Briefly, 1 M lanthanum chloride and 2 M potassium dihydrogen phosphate were added to the solution of total cellular proteins (0.5 - 2 mg) to precipitate phosphoproteins as described [6 (link)]. After the washing steps, the phosphoproteins were eluted from the pellet using a mixture of 25% 4 M Imidazole and 75% sample buffer (8 M urea, 2 M thiourea, 2% CHAPS, 1% DTT in water) and purified by cold acetone/20% TCA in water solution. Phosphoproteins were finally resuspended in IEF buffer [8 M urea, 2% w/v CHAPS, 0.5% ampholine (pH 3-10), 18 mM DTT, 0.002% w/v bromophenol blue (BBP)].
In the second approach, phosphoproteins of 4 PBMCs samples, different from those used for lanthanum ions precipitation, were enriched by Pro-Q® Diamond Phosphoprotein Enrichment kit of Invitrogen. Phosphoproteins were isolated according to the manufacturer’s protocol for 0.5-1 mg of protein extract. All samples were run at least in duplicate.

Acetonitrile (ACN), acetone, trifluoroacetic acid (TFA), trichloroacetic acid (TCA), DL-dithiothreitol (DTT), iodoacetamide (IAA), glycine, EDTA, Tris, endonuclease, phosphoprotease and protease inhibitors, lanthanum chloride, potassium dihydrogen phosphate, Coomassie Blue G-250, imidazole, alkaline phosphatase were purchased from Sigma (Sigma Aldrich St.Louis, MO, USA); urea, CHAPS, SDS, glycerol, acrylamide, ampholine, and Ficoll-Paque™ were purchased from GE healthcare (Uppsala, Sweden); Agarose, Pro-Q® Diamond Phosphoprotein Enrichment kit, Pro-Q® Diamond dye, SYPRO® Ruby and PeppermintStick™ Phosphoprotein Molecular Weight Standards were from Invitrogen™ (Carlsbad, CA); piperazine di-acrylamide (PDA), TEMED, Bio Rad Protein Assay, IPG strips, were from Bio-Rad Laboratories (Hercules, CA). Trypsin (sequencing grade modified) was from Promega (Madison, Wisconsin, USA). All solvents used were Ultra-Resi-Analyzed grade.