The cells lysates were obtained with RIPA buffer (50 mM Tris, PH 8.0,150 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate) containing proteinase inhibitors (1% inhibitors cocktail and 1 mM PMSF) (Roche Applied Science, Germany) for 10 min in ice and centrifuged at 12,000 g for 15 min at 4 °C. The samples were separated by 12% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% non-fat milk, the membranes were incubated with primary antibody at 4 °C overnight. Next, the membranes were washed with TBST buffer for 3 times and incubated with peroxidase-conjugated secondary antibody for 1 h at room temperature. After washing with TBST buffer for 3 times again, the membranes were visualized with an ECL chemiluminescent detection system (Bio-rad, USA).
Inhibitors cocktail
Manufactured by Roche
Sourced in Germany
The Inhibitors cocktail is a laboratory product from Roche. It is designed to inhibit a range of enzymes and proteins that may interfere with experimental procedures. The specific composition and function of this product are as follows: The Inhibitors cocktail contains a combination of chemical compounds that act as inhibitors for various proteases, phosphatases, and other enzymes. This product is intended to help maintain the integrity of samples and prevent unwanted modifications or degradation during research and analytical processes.
Automatically generated - may contain errors
4 protocols using inhibitors cocktail
1
Western Blot Analysis with RIPA Lysis
2
Integrin Crosslinking Analysis
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Cells were seeded onto pFN-coated glass coverslips and spun and non-spun cells were incubated with 1 mM 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP; Thermo Scientific, Madrid, Spain) during 15 min at 4°C. Quenching was carried out with 50 mM Tris, pH 7.4 for 15 min at RT and cells were extracted with 20 mM Tris, pH 7.4, 0.1% SDS and proteinase inhibitors (Inhibitors cocktail, Roche , Barcelona, Spain). Cell lysates were collected and coverslips were thoroughly washed with 20 mM Tris, pH 8.5 followed by incubation with 20 mM Tris, pH 8.5, 0.1% SDS and 25 mM DTT for 1 hr at 37°C to break the crosslinks. The whole crosslinked fractions and the cell lysates were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Western-blots were analyzed with ImageJ and the levels of crosslinked integrins were calculated as the relation between the crosslinked and the total integrin fractions (cell lysates + crosslinked fraction).
3
Western Blot Analysis of Protein Lysates
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The cell lysates were prepared using RIPA buffer (50 mM Tris, PH 8.0, 150 mM NaCl, 0.1% SDS, 1% NP40 and 0.5% sodium deoxycholate) containing proteinase inhibitors (1% inhibitors cocktail and 1 mM PMSF) (Roche Applied Science, Germany) for 10 min in ice and centrifuged at 12000g for 15 min at 4°C. The samples were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking with 5% bovine serum albumin, the membranes were incubated with primary antibody at 4°C overnight. Next, the membranes were washed with TBST buffer for 3 times and incubated with peroxidase-conjugated secondary antibody for 1h at room temperature. After washing with TBST buffer for 3 times again, the membranes were visualized by using an ECL chemiluminescent detection system (Bio-rad, USA).
4
Isolation of Mitotic Chromosome Chromatin
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Either LAP, LAP-Aurora B, LAP-Survivin or LAP-Borealin expressing HeLa cells (3x109) were arrested with colcemid for 16-18 hours were harvested and mitotic chromosomes were purified as described (Paulson, 1982) . Mitotic chromosomes were resuspended in 40ml
MNase buffer (20mM HEPES, pH7.7; 20mM KCl; 5mM β-mercaptoethanol; 1xProtease
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Inhibitors cocktail (EDTA free, Roche), 20mM β-glycerophosphate,1mM Sodium orthovanadate, 3mM CaCl2, 250 mM NaCl , and 0.1% Digitonin) and digested with MNase (Roche, 150u ml-1)
for 1 hour at room temperature. After digestion extracts were supplemented with 50 mM NaF and clarified by centrifugation at 12,000g for 20 minutes at 4°C. The supernatants were used as starting materials for LAP purifications that are described in the Figure 1-figure supplement 1.
MNase buffer (20mM HEPES, pH7.7; 20mM KCl; 5mM β-mercaptoethanol; 1xProtease
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
Inhibitors cocktail (EDTA free, Roche), 20mM β-glycerophosphate,1mM Sodium orthovanadate, 3mM CaCl2, 250 mM NaCl , and 0.1% Digitonin) and digested with MNase (Roche, 150u ml-1)
for 1 hour at room temperature. After digestion extracts were supplemented with 50 mM NaF and clarified by centrifugation at 12,000g for 20 minutes at 4°C. The supernatants were used as starting materials for LAP purifications that are described in the Figure 1-figure supplement 1.
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