Readyprep 2 d cleanup kit

The harvested cells were re-suspended and disrupted in a lysis buffer composed of 7 M urea, 2 M thiourea, 4% w/v 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate, 20 mM tributyl phosphate, and 0.2% Bio-lyte (pH 3–10), and a protease inhibitor cocktail (Roche Diagnostics Ltd, Mannheim, Germany). DNAse I and RNAse A were added to the lysate at final concentrations of 1 mg mL⁻¹ and 0.25 mg mL⁻¹, respectively. After disruption, the protein solution was separated from the cell debris by centrifugation (12,000 × g, 5 min, 4 °C). The crude protein extracts were further purified using a Ready Prep 2-D Cleanup Kit (Bio-Rad Laboratories, USA) before undergoing a reductive alkylation reaction. The protein concentration was determined using a 2-D Quant Kit (GE Healthcare, USA). Digestion and labeling were performed according to the manufacturer’s protocol (Applied Biosystems). Briefly, 100 μg of total protein in the cell fraction was reduced and alkylated, then digested overnight at 37 °C with trypsin and labeled with iTRAQ-reagents (Applied Biosystems) as follows: w0d, iTRAQ reagent 115; w1d, iTRAQ reagent 116; w3d, iTRAQ reagent 117; w5d, iTRAQ reagent 118; and w7d, iTRAQ reagent 121.

Adult mice (10–15 mo old) were placed in a CO₂ atmosphere and then decapitated. After carefully removing the skull, the hippocampus was dissected out, frozen rapidly in liquid nitrogen and stored at −80°C until processing. ∼0.5 mg of hippocampal tissue from the Igf-I^Ctrl (or Igf-I control) or Igf-I^Δ/Δ (or Igf-I conditional knockout -cKO- mice) was homogenized in lysis buffer containing 7 M urea, 2 M thiourea, and 50 mM DTT. The homogenates were centrifuged at 100,000g for 1 h at 15°C. Protein precipitation was performed using the ReadyPrep 2-D Cleanup Kit (Bio-Rad) following the manufacturer’s instructions. Then, protein concentration was measured using the Bradford assay kit (Bio-Rad).

The proteome of wildtype and mutant organisms were extracted from independent replicates and the protein samples were separated into two portions, one of which was used for 2D gel experiment and the other for quantitative shotgun proteome analysis. The comprehensive protein expression data was compared between replicates of the same group of wildtype and mutants and then by comparing wildtype with the mutants. Supplementary Figure 1 has the workflow of sample preparation, LC-MS/MS analysis and proteins quantitation of E. chaffeensis wildtype and mutants organisms. Purified cell-free Ehrlichia were resuspened in lysis buffer containing 8M urea, 2M thiourea, 4% CHAPS, and protease inhibitors (Roche, Indianapolis, IN). Samples were sonicated on ice for 30 s using sonic dismembrator (Fisher Scientific, Hampton, NH) and centrifuged at 15,000x g for 15 min at 4°C. Proteins were precipitated and purified using a Readyprep 2D cleanup kit (BioRad, Hercules, CA) and then quantified using a detergent-compatible Bradford protein assay kit (BioRad, Hercules, CA). The proteins were reduced and alkylated using 10 mM DTT and 40 mM IAA, respectively and diluted 7 times with 100 mM TEAB.

Tissue specimens were subjected to membrane protein enrichment using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific, Waltham, MA, USA) which applies a mild detergent-based selective extraction protocol to enrich integral membrane proteins and membrane-associated proteins. The extraction was performed essentially according to manufacturer’s instructions for hard tissue, except that buffer volumes were adjusted for the samples depending on the weight. The membrane-enriched fraction was used for all further analysis of the samples in this study. The membrane-enriched fraction from each of the tissue samples was cleaned up using the ReadyPrep 2D Clean-Up Kit (Bio-Rad, CA, USA), according to manufacturer’s instructions. The cleaned protein pellets were resuspended in a buffer containing 6 M urea, 2 M thiourea, and 10 mM Tris, pH 8.5, and assayed for protein concentration using the QuickStart Bradford Protein Assay (Bio-Rad). Fifteen micrograms of protein were suspended with 50 mM ammonium bicarbonate, and protein digestion was carried out as previously described [22 (link)].

Proteins were cleaned using the ReadyPrep 2D cleanup kit (Bio-Rad) following the manufacturer’s instructions.

Amicon Ultra-15 10K Centrifugal Filter Devices (Millipore) were used to reduce the sample volume to approximately 200 µL (4700 g, 4 °C). This final volume was washed and desalted using the Bio-Rad ReadyPrep 2-D Cleanup Kit. Finally, the dried protein samples were solubilized in 100 µL labelling buffer (7 M urea, 2 M thiourea, 2% (w/v) CHAPS, 30 mM Tris), and the Bradford protein assay with bovine serum albumin as standard was used to determine the protein concentrations.

Amoebic protein was obtained from an axenic culture of Entamoeba histolytica HM-1:IMSS. Briefly, trophozoites were harvested by centrifugation and resuspended in buffer A (0.05 M Tris-HCl pH 6.8, added with 5% Triton X-100 and a protease inhibitor cocktail containing 1 mM phenylmethylsulfonylfluoride (Sigma, Chemical Co.), 2 μM leupeptin, and 5 mM N-ethylmaleimide (Sigma, Chemical Co.)). The trophozoites were lysed in a Teflon glass Potter homogenizer coupled to a drill at 3000 rpm with 80 up and down strokes in an ice bath. Homogenate was centrifuged for 30 min at 12,000 rpm at 4°C and the obtained pellet was suspended in 0.36 mL of buffer A and 1.44 mL of OFFGEL buffer stock (1.25X) (Agilent Technologies, St. Clara, USA). Later, proteins were separated according to their isoelectric point (pI) using the Agilent 3100 OFFGEL fractionator (Agilent Technologies, St. Clara, USA), pH gradient ranging from 3 to 10. One aliquot of each batch, was screening in 2100 BioAnalyzer (Agilent Technologies, St. Clara, USA) to identify a single protein fraction; finally, the fraction corresponding to pH 4.6–5.4, which contained a 46 kDa protein, was cleaned using the ReadyPrep-2D CleanUp kit (BioRad) to eliminate salts and buffers that could interfere in the functionalization procedure. The protein was quantified using the Bradford assay [14 (link)] and stored at −70°C until use.