Samples were processed as described by (Hornburg et al., 2014 (link)). In brief, cell pellets were washed with PBS and lysed in 4% SDS, 10 mM HEPES (pH 8), 10 mM DTT. Cell pellets were heat-treated at 95°C for 10 min and sonicated at 4°C for 15 min (level 5, Bioruptor, Diagenode). Alkylation was performed in the dark for 30 min by adding 55 mM iodoacetamide (IAA). Proteins were precipitated overnight with acetone at −20°C and resuspended the next day in 8 M Urea, 10 mM HEPES (pH 8). A two-step proteolytic digestion was performed. First, samples were digested at room temperature (RT) with LysC (1:50, w/w) for 3h. Then, they were diluted 1:5 with 50 mM ammoniumbicarbonate (pH 8) and digested with trypsin (1:50, w/w) at RT overnight. The resulting peptide mixtures were acidified and loaded on C18 StageTips (Rappsilber et al., 2007 (link)). Peptides were eluted with 80% acetonitrile (ACN), dried using a SpeedVac centrifuge (Eppendorf, Concentrator plus, 5305 000.304), and resuspended in 2% ACN, 0.1% trifluoroacetic acid (TFA), and 0.5% acetic acid. For deeper proteome analysis a peptide library was built. For this, peptides from naive and activated T cells were separated according to their isoelectric point on dried gel strips with an immobilized pH gradient (SERVA IPG BlueStrips, 3-10 / 11 cm) into 12 fractions as described by Hubner et al., 2008 (Hubner et al., 2008 (link)).
Ipg bluestrips
Manufactured by Serva Electrophoresis
Sourced in Germany
IPG BlueStrips are pre-cast immobilized pH gradient (IPG) gel strips used in isoelectric focusing, a key step in two-dimensional gel electrophoresis (2D-PAGE) for protein separation and analysis. The strips provide a stable and reproducible pH gradient across the length of the strip, allowing for the separation of proteins based on their isoelectric point.
Automatically generated - may contain errors
3 protocols using ipg bluestrips
1
Proteome Sample Preparation Workflow
2
Myelin Protein Solubilization and Isoelectric Focusing
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A volume equivalent corresponding to 100 µg myelin protein was mixed with the same volume of rehydration buffer I (7 M urea, 2 M thiourea, 2% (w/v) ASB-14, 40 mM DTT, 1% (v/v) Servalyte ampholytes pH 3–10 (Serva)) and solubilized by short sonication and gentle shaking. The sample was filled up to 130 µL with rehydration buffer II (7 M urea, 2 M thiourea, 2% (w/v) ASB-14, 20 mM DTT, 0.5% (v/v) Servalyte ampholytes pH 3–10 (Serva)), solubilized as above, centrifuged (2 min 16,000× g), and the supernatant subjected to IEF in immobilized pH gradients (IPG BlueStrips, 7 cm, pH 3–10 or 3–12, Serva) in a Protean i12 IEF System (BioRad, Munich, Germany). During active rehydration at 50 V, 20 °C for ~16 h, current was limited to 70 µA. After 6 h active rehydration, paper wigs moisturized with distilled water were placed at both electrode ends underneath the IPG strips. For isoelectric focusing a step-gradient protocol was set according to the manufacturer’s instructions (1: hold at 150 V for 1 h; 2: hold at 300 V for 1 h; 3: ramp to 1000 V in 1 h; 4: ramp to 3000 V in 2 h; 5: hold at 3000 V for 2 h) at 20 °C.
3
Two-Dimensional Electrophoresis of Co-IP Eluate
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The Hoefer IEF100 isoelectric focusing unit (Hoefer, San Francisco, CA, USA) was used to perform the first-dimension electrophoresis. IPG Blue strips (7 cm, pH 3–10 and pH 5–8, linear), ampholytes (ServalytTM, pH 3–10 and pH 5–8) and HPE IPG Overlay were purchased from Serva Electrophoresis (Heidelberg, Germany). Preparation of solutions and rehydration of strip gels were performed according to the IEF100 manual. Co-IP eluate (about 10 µg protein) was applied by cup loading after full denaturation with 2DE compatible buffer. The IEF100 preprogrammed protocol for 7 cm IPGs applying constant watt conditions was used. A two-step equilibration of IPG strips was performed before the second dimension SDS-PAGE. A minigel system (Hoefer, San Francisco, CA, USA) and 10% polyacrylamide gels were employed for SDS-PAGE. The IPG strip was placed on the top of the SDS gel. A piece of filter paper loaded with prestained peqGold Protein Marker V (Peqlab, Erlangen, Germany) was placed on the alkaline end of the IPG strip, and an overlay of hot agarose was used to seal the strip. SDS gels were transferred and subjected to silver staining. Pierce Silver Stain for Mass Spectrometry (Thermo Scientific, Rockford, IL, USA) was used according to the kit’s manual. Image acquisition of stained gels was performed using a scanner (Epson V750, Tokyo, Japan).
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