Human apotransferrin (Sigma-Aldrich, St Louis, MO) was labeled according to the method of Bates and Wernicke (Bates and Wernicke, 1971 (link)) as described before (Goralska et al., 1998 (link)). LEC were preincubated for 1 h in serum free MEM (Mediatech) under normoxic conditions to remove transferrin bound to the membrane and then labeled with 59FeTf (70–180 ng of Fe) in 1ml fresh, serum-free MEM. LEC were exposed to hypoxia (0.5% O2; 5% CO2) or incubated under normoxic conditions for 6 or 24 h and lysed with 10 mM Tris/HCl buffer containing 15% sucrose and 6 μl/ml of Protease Inhibitor Cocktail. Lysates were centrifuged at 15,000 g. The radioactivity of collected supernatants was measured in a gamma counter 1480 Wallac Wizard (Wallac, Turku, Finland). The supernatants were subsequently precipitated with 50% acetone (10 min on ice) and proteins were pelleted at 15 000 g. Pellets were dissolved in non-denaturing PAGE loading buffer and proteins were separated on native 8% PAGE. The dried gels were exposed to film and quantified in a radioactivity detector (Instant Imager, Packard-Canberra, Rockville, MD). The radioactive bands of ferritin were identified based on their mobility in comparison to Kaleidoscope Protein Standards (Bio-Rad, Richmond, CA).
Kaleidoscope protein standards
Manufactured by Bio-Rad
Sourced in United States
Kaleidoscope protein standards is a set of pre-stained molecular weight marker proteins for estimating the molecular weights of proteins by SDS-PAGE. It provides a visually distinctive pattern of bands for easy identification of protein sizes.
Automatically generated - may contain errors
Lab products found in correlation
Human apo transferrin, by Merck Group (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Enhanced chemiluminescence reagent, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Proteintech (1 mentions)
Anti hcv core 1b antibody, by Abcam (1 mentions)
4 protocols using kaleidoscope protein standards
1
Labeling and Analysis of Cellular Ferritin
2
Protein Visualization via ECL Detection
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This was performed as described previously [14 (link)]. The protein bands were visualized using the ECL protein detection system (Amersham, USA). In all cases the apparent molecular masses were estimated using Kaleidoscope protein standards (Bio Rad, USA).
3
HCV Core Protein Quantification
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For this assay, total proteins were extracted using a radioimmunoprecipitation assay lysis buffer and quantitated using bicinchoninic acid protein assay. The lysates were mixed with an equal volume of 2× loading buffer and boiled for 5 min. Then, 50 μg of proteins was subjected to electrophoresis on 10% SDS-PAGE gels (1.0 mm thick × 15 cm long), along with 15 μl of Kaleidoscope protein standards (Bio-Rad), and transferred to polyvinylidene difluoride membranes (Millipore). After blocking with 5.0% bovine serum albumin in tris-buffered saline and Tween 20, target proteins on membranes were probed at 37°C for 1 hour with anti–HCV Core 1b antibody (1:1000 dilution; Abcam). β-Actin was used as a loading control for analysis and was probed by monoclonal mouse anti-actin antibody (1:1000 dilution; Abcam). After the final wash, the signal was developed with corresponding horseradish peroxidase–conjugated secondary antibody (1:5000; Proteintech). Finally, protein bands were visualized on an x-ray film using enhanced chemiluminescence reagents (Millipore).
4
Western Blot Analysis of STAT1 and pSTAT1
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Cell lysates were prepared in 1X boiling mix (1% SDS, 15% glycerol, 1% β-mercaptoethanol, 60 mM sodium phosphate, pH 6.8) and heated at 100 °C for 2 min. The protein sample was separated by SDS-PAGE. After SDS-PAGE, the proteins were transferred on to PVDF membranes using the mini blotting apparatus (BioRad, USA), after which the membranes were washed with PBSA and blocked for 18 h at 4 °C in PBSA containing 1% BSA and 0.05% Tween 20. The membrane was incubated with the respective specific primary antibody to STAT1 and pSTAT1, followed by the appropriate anti-mouse or anti-rabbit IgG (whole molecule) peroxidase conjugate (Sigma, USA). The protein bands were visualized using the ECL protein detection system (Amersham, Buckinghamshire, UK). In all cases, the apparent molecular masses were estimated using Kaleidoscope protein standards (BioRad, USA).
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