Whole cell lysates were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with Complete Mini and PhosSTOP tablets (Roche). Total protein levels were determined via Bio-Rad protein assay. Equal protein amounts were electrophoresed on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P; Millipore). The following primary antibodies were used: RRM2, RRM1, p53R2, GAPDH, Involucrin (Santa Cruz); Chk1 (Abcam); phospho-Chk1 Ser345, E2F1 (Cell Signaling Technologies). Secondary antibodies used were: HRP conjugated anti-goat (Santa Cruz), HRP conjugated anti-rabbit (Cell Signaling Technologies), and HRP conjugated anti-mouse (GE Life Sciences). Clarity Western enhanced chemiluminescence (ECL) blotting substrate (Bio-Rad) was used to detect antibody binding.
Clarity western enhanced chemiluminescence ecl blotting substrate
Manufactured by Bio-Rad
Sourced in United Kingdom
Clarity Western enhanced chemiluminescence (ECL) blotting substrate is a laboratory product designed for the detection and analysis of proteins in Western blot experiments. It provides a chemiluminescent signal that can be captured and quantified using imaging equipment.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (1 mentions)
Hrp conjugated anti mouse, by GE Healthcare (1 mentions)
Hrp conjugated anti goat, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated anti rabbit, by Cell Signaling Technology (1 mentions)
Iblot, by Thermo Fisher Scientific (1 mentions)
Westernbreeze blocker diluent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Roche (1 mentions)
Ibright 1500, by Thermo Fisher Scientific (1 mentions)
I block, by Thermo Fisher Scientific (1 mentions)
An antibody against β actin, by Abcam (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Complete mini protease inhibitor cocktail, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
4 protocols using clarity western enhanced chemiluminescence ecl blotting substrate
1
Western Blot Analysis of DNA Repair Proteins
2
Western Blot Analysis of Protein Expression
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After electrophoresis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes using the iBlot (Invitrogen) dry blotting system. Polyclonal rabbit anti-StK and anti-StpA antibodies were used at recommended dilutions of 1/2,000 and 1/1,000, respectively (8 (link)). A Bis-Tris detergent-buffered saline solution with Hammersten casein was used for the block and antibody dilutions (Invitrogen; Western Breeze blocker/diluent). Goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Invitrogen) was used at a 1/10,000 dilution, and the proteins were detected with Clarity Western enhanced chemiluminescence (ECL) blotting substrate (Bio-Rad).
3
Quantifying ArlB Protein in Biofilm and Planktonic Cells
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To assay the production of ArlB in planktonic cells and biofilm, S. acidocaldarius MW001 and mutant strains were grown as for RNA isolation. Biofilm was scraped off the petri dishes and resuspended in 1 mL growth medium. The OD600s of cells from biofilm and planktonic cultures were determined. Cells were pelleted at 2,400 × g in a tabletop centrifuge for 10 min and resuspended to a theoretical OD of 10 in 1× SDS loading dye. The whole-cell samples were separated on SDS-PAGE and blotted on polyvinylidene difluoride (PVDF) membrane (Roche). The membrane was blocked with I-Block (Thermo Fisher Scientific) and incubated in primary antibody against ArlB (Eurogentec) overnight at 4°C. Afterwards, the membrane was incubated in secondary goat anti-rabbit antibody coupled to horseradish peroxidase (HRP) overnight at 4°C. Chemiluminescent signals were recorded with IBright 1500 (Invitrogen, Thermo Fisher Scientific) using Clarity Western ECL enhanced chemiluminescence blotting substrate (Bio-Rad).
4
Western Blot Analysis of LEKTI in Keratinocytes
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Keratinocytes were lysed in protein extraction buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Nonidet P-40, and 5 mM EDTA) containing proteinase inhibitors cocktail (cOmplete Mini protease inhibitor cocktail, Roche) and phosphatase inhibitors cocktail (PhosSTOP, Roche). For each sample, 20 μg of total protein was resolved on NuPAGE 4%–12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and electrotransferred onto nitrocellulose membranes (Invitrogen).
For LEKTI analysis, blots were probed with a polyclonal anti-LEKTI antibody38 (link) at 1:1,000 dilution, and cell immortalization was assessed with an anti-E7 monoclonal antibody (Santa Cruz Biotechnology. Dallas, TX, USA) at 1:1,000 dilution. An antibody against β-actin (Abcam, Cambridge, UK) was used as loading control. Visualization was performed by incubating the membrane with horseradish peroxidase (HRP)-conjugated anti-immunoglobulin G (IgG) rabbit antibody (Amersham, Burlington, MA, USA) or anti-IgG mouse antibody (Jackson ImmunoResearch Laboratories. Cambridge, UK) at 1:5,000 dilution and Clarity western ECL (enhanced chemiluminescence) blotting substrate (Bio-Rad).
For LEKTI analysis, blots were probed with a polyclonal anti-LEKTI antibody38 (link) at 1:1,000 dilution, and cell immortalization was assessed with an anti-E7 monoclonal antibody (Santa Cruz Biotechnology. Dallas, TX, USA) at 1:1,000 dilution. An antibody against β-actin (Abcam, Cambridge, UK) was used as loading control. Visualization was performed by incubating the membrane with horseradish peroxidase (HRP)-conjugated anti-immunoglobulin G (IgG) rabbit antibody (Amersham, Burlington, MA, USA) or anti-IgG mouse antibody (Jackson ImmunoResearch Laboratories. Cambridge, UK) at 1:5,000 dilution and Clarity western ECL (enhanced chemiluminescence) blotting substrate (Bio-Rad).
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