Zoom ipgrunner system

To concentrate proteins and remove impurities such as salts or detergents that may interfere with 2-D SDS-PAGE separation, the extracts were precipitated with the ProteoExtract protein precipitation kit (Calbiochem Darmstadt, Germany). The pure cytosol proteins were solubilized in sample rehydration buffer (8 M urea, 2% CHAPS, 0.5% ZOOM Carrier Ampholytes). 2-DE was carried out in a ZOOM IPGRunner System (Invitrogen, Carlsbad, CA). Immobilized pH gradient (IPG) strips 7 cm in length with pH ranges of 5–8 were rehydrated overnight in 155 μL of the sample rehydration buffer containing traces of bromophenol blue, 20 mM DTT, and 100 μg protein. The first dimension was performed using a multistep protocol (25 min at 200 V, 15 min at 450 V, 15 min at 750 V, and 100 min at 2,000 V). At the end of isoelectric focusing, each strip was equilibrated in two steps of 15 min in 5 mL of Laemmlisample buffer supplemented with 10 mg/mL DTT instead of β-mercaptoethanol, and 40 mg/mL iodoacetamide, respectively. The second-dimension SDS-PAGE and Western blot were performed as described below. The preparative gel was stained with Coomassie blue in accordance with the manufacturer's instructions for antigen identification.

We used the Zoom IPGRunner system (Invitrogen) to separate proteins in two dimensions. We isolated the HSPCs of young male and female C57BL/6J mice (four to eight months of age) and lysed them according to the manufacturer’s instructions with urea, CHAPS, dithiothreitol (DTT) and ampholytes. CyDyes DIGE fluors (minimal dyes) were used according to the vendor’s instructions (Amersham) with fluorophores Cy3 and Cy5 for post-lysis labelling to ensure that only 1–2% of lysines were labelled in a given protein. Labelling intensities were measured with a Typhoon FLA 9000 scanner and quantified with DeCyder 7.0 and ImageQuant software (GE Healthcare). We normalized the total protein abundance based on protein size and lysine concentration for spots with known identity by MS/MS. PPIA quantity was identical in two independent experiments using different fluorophores.

To show active MMP9 in the tissue 2D zymography was used (a technique pioneered in our laboratory 19 (link)). Briefly, the tissue was minced in cocadylic acid and the tissue extract was mixed with ampholyte (pH 3–10; Invitrogen) to prepare the sample rehydration buffer as per manufacturer’s protocol. The zoom IPG strips (pH 3–10) were soaked in sample rehydration buffer in the zoom Immobilized PH Gradient runner cassette, overnight and isoelectrofocusing was done in the zoom IPG runner system (Invitrogen) using the step voltage program as per manufacturer’s instructions. The strip was placed on 10% SDS-PAGE gel prepared with 2% gelatin and electrophoresed until the dye passed out. The gel was washed three times in 2.5% Triton X-100 for 20 min. each to remove SDS and incubated in activation buffer (5 mmol/l Tris HCl- pH 7.4, 0.005% v/v Brij-35, and 1 mmol/l CaCl₂) for 24 hrs at 37°C. The gel was stained in commasie and destained to observe a clear zone against blue background as a result of proteolytic acitivity of MMPs for gelatin, along with controls. The gels were imaged with gel documentation system (Bio-Rad, Hercules, CA, USA) and data were analysed using image lab software (Bio-Rad).