Western blot was performed as described previously [12 (link)]. Briefly, total (25 µg), cytoplasmic (25 µg) or nuclear (30 µg) proteins were separated by SDS-PAGE under reducing conditions and electrotransferred onto PVDF membranes which were probed overnight at 4 °C or for 2 h at room temperature with the primary antibodies listed in Table 1 and then with anti-rabbit (dilution 1:20000; NIF1317, lot9465473, GE Healthcare, Chalfont St. Giles, UK) or anti-mouse (dilution 1:20000; NIF1316, lot6963606, GE Healthcare, Chalfont St. Giles, UK) alkaline phosphatase-conjugated secondary antibodies. Mouse monoclonal anti-β-tubulin I and rabbit polyclonal anti-lamin B1 were used as loading controls of total and cytoplasmic extracts and of nuclear extracts, respectively. Immune complexes were detected with Enhanced ChemiFluorescence reagent (GE Healthcare) in the imaging system ThyphoonTM FLA 9000 (GE Healthcare). Image analysis was performed with TotalLab TL120 software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).
Enhanced chemifluorescence reagent
Manufactured by GE Healthcare
The Enhanced ChemiFluorescence reagent is a laboratory product designed to enhance chemifluorescence detection in various analytical applications. It is intended to improve the sensitivity and performance of chemifluorescence-based assays, but its specific intended use is not elaborated upon.
Automatically generated - may contain errors
Lab products found in correlation
Fviia, by Enzyme Research (3 mentions)
Fixaβ, by Enzyme Research (3 mentions)
α thrombin, by Enzyme Research (3 mentions)
Nif1317, by GE Healthcare (2 mentions)
Nif1316, by GE Healthcare (2 mentions)
Thyphoontm fla 9000, by GE Healthcare (2 mentions)
Anti rabbit or anti mouse alkaline phosphatase conjugated secondary antibodies, by GE Healthcare (2 mentions)
Hirudin, by Diapharma (1 mentions)
Thrombin calibrator, by Diagnostica Stago (1 mentions)
Innovin, by Siemens (1 mentions)
Phosphatidylserine, by Avanti Polar Lipids (1 mentions)
Phosphatidylethanolamine, by Avanti Polar Lipids (1 mentions)
Phosphatidylcholine pc, by Avanti Polar Lipids (1 mentions)
Thyphoon fla 9000, by GE Healthcare (1 mentions)
Sc 7884, by Santa Cruz Biotechnology (1 mentions)
Mab9502, by R&D Systems (1 mentions)
Nzycolour protein marker 2, by NZYTech (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Imagequant tl software, by GE Healthcare (1 mentions)
5 protocols using enhanced chemifluorescence reagent
1
Western Blot Analysis of Cellular Proteins
2
Recombinant FVIII Coagulation Assay
3
Western Blot Analysis of IL-1β and NOS2
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Total cell extracts were prepared and western blot was performed as described before54 (link). Briefly, total (25 µg for Raw 264.7 cell line and 20 µg for human chondrocytes) proteins were separated by SDS-PAGE under reducing conditions and electrotransferred onto PVDF membranes. These were probed overnight at 4 °C or for 2 h at room temperature with rabbit polyclonal antibody against IL-1β (dilution 1:500; sc-7884, Santa Cruz Biotechnology, INC., Texas, USA) or mouse monoclonal antibody NOS2 (dilution 1:500; MAB9502, R&D Systems, Minneapolis, MN, USA) and then with anti-rabbit or anti-mouse alkaline phosphatase-conjugated secondary antibodies (dilution 1:20000; GE Healthcare, Chalfont St. Giles, UK) for 1 h at room temperature. Immune complexes were detected with Enhanced ChemiFluorescence reagent (GE Healthcare) in the imaging system Thyphoon FLA 9000 (GE Healthcare). The membranes were reprobed with a mouse monoclonal anti-β-Tubulin I antibody (Sigma-Aldrich Co.), diluted at 1:20000, as a loading control, for 1 h at room temperature. Image analysis was performed with TotalLab TL120 software (Nonlinear Dynamics Ltd).
4
Western Blot Analysis of Proteins
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Western blot was performed as described previously [13 (link)]. Briefly, total (25 µg), cytoplasmic (25 µg) or nuclear (30 µg) proteins were separated by SDS-PAGE under reducing conditions. A commercial mixture of 12 purified pre-stained proteins (NZYColour Protein Marker II, NZYTech, Lisbon, Portugal) was run in each gel to allow for confirmation of the apparent molecular weight of the proteins of interest. The proteins were then electrotransferred onto PVDF membranes (Immobilon®—P, Merck Millipore Ltd.) which were probed overnight at 4 °C or for 2 h at room temperature with the primary antibodies indicated in Table 1 and then with anti-rabbit (dilution 1:20,000; NIF1317, lot9465473, GE Healthcare, Chalfont St. Giles, UK) or anti-mouse (dilution 1:20,000; NIF1316, lot6963606, GE Healthcare, Chalfont St. Giles, UK) alkaline phosphatase-conjugated secondary antibodies. Mouse monoclonal anti-β-Tubulin I and rabbit polyclonal anti-Lamin B1 were used as a loading controls of total and cytoplasmic extracts and of nuclear extracts, respectively. Immune complexes were detected with Enhanced ChemiFluorescence reagent (GE Healthcare) in the imaging system ThyphoonTM FLA 9000 (GE Healthcare). Image analysis was performed with TotalLab TL120 software (Nonlinear Dynamics Ltd., Newcastle upon Tyne, UK).
5
Western Blot Analysis of Protein Signaling
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Total and cytoplasmic extracts were prepared as described before (Goldring and Otero, 2011) . Proteins were separated by SDS-PAGE under reducing conditions and electrotransferred onto PVDF membranes. These were probed overnight with the following primary antibodies: mouse monoclonal anti-human iNOS (R&D systems, Minneapolis, MN), mouse polyclonal anti-human phospho-IκB-α or rabbit polyclonal anti-human IκB-α, antihuman phospho-JNK, anti-human phospho-p38 or anti-human phospho-ERK1/2 (Cell Signaling Technology, Inc., Danvers, MA) and then with anti-rabbit or anti-mouse alkaline phosphataseconjugated secondary antibodies (GE Healthcare, UK). Mouse antihuman β-Tubulin monoclonal antibody was used to detect β-Tubulin as a loading control. Enhanced ChemiFluorescence reagent (GE Healthcare) was used to detect immune complexes. Image analysis was performed with ImageQuant TL software (GE Healthcare). The results presented are the normalized ratio between the intensities of the bands corresponding to the protein of interest and to the protein used as loading control.
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