Cultured cortical neurons or HEK cells were rinsed with cold aCSF and collected in 200 μL lysis buffer [50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% Sodium deoxycholate (SDOC) and 1% sodium dodecyl sulfate (SDS)] containing mini cOmplete protease inhibitors (Hoffmann-La Roche Grenzacherstrasse, Basel, Switzerland). The high SDS concentration was applied to avoid conventional protein–protein interaction. Lysates were further solubilized by sonication and 10 min incubation on ice followed by centrifugation for 10 min at 13 000 g. Supernatant was adjusted to 500 μL with lysis buffer and incubated 3 h on rotation at 4°C with 1μL antibodies against XIAP and protein A-Sepharose beads (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunocomplexes were washed three times with ice-cold lysis buffer without SDS (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP40, 1% SDOC), resuspended in 2X Laemmli buffer and denatured at 95°C for 10 min.
Minicomplete protease inhibitors
Manufactured by Roche
Sourced in Switzerland
MiniComplete protease inhibitors are a set of reagents designed to inhibit the activity of proteases, which are enzymes that break down proteins. These inhibitors are commonly used in various laboratory applications, such as protein extraction and purification, to prevent proteolytic degradation and maintain the integrity of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Phosstop phosphatase inhibitor, by Roche (5 mentions)
Anti total enos, by BD (2 mentions)
Clone c4, by Santa Cruz Biotechnology (2 mentions)
Anti p s1177 enos, by BD (2 mentions)
Horse radish peroxidase conjugated donkey anti mouse or anti rabbit secondary antibody, by Jackson ImmunoResearch (2 mentions)
Anti total enos, by Santa Cruz Biotechnology (2 mentions)
Clone 3 enos nos type 3, by Santa Cruz Biotechnology (2 mentions)
Anti β actin, by Santa Cruz Biotechnology (2 mentions)
Anti piezo1, by Proteintech (2 mentions)
Mouse anti human β actin, by Santa Cruz Biotechnology (2 mentions)
Protein a sepharose beads, by Santa Cruz Biotechnology (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
G box chemi xt4, by Syngene (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Fastap, by Thermo Fisher Scientific (1 mentions)
Fastap buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti human cd31, by Abcam (1 mentions)
Phosphatase inhibitor cocktail, by Roche (1 mentions)
Vinculin, by Cell Signaling Technology (1 mentions)
Fabp5, by Cell Signaling Technology (1 mentions)
14 protocols using minicomplete protease inhibitors
1
Immunoprecipitation of XIAP Protein
2
Labeling and Purification of RAD51 in MEFs
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DAz-2, synthesized as previously described [58 (link)] was prepared in DMSO at a final concentration of 250 mM and stored at −20 °C. p-Biotin was prepared in DMSO at a final concentration of 5 mM and stored at −80 °C. Prdx1+/+ and Prdx1−/− MEFs expressing WT FLAG-RAD51 or Prdx1+/+ MEFs expressing C137S, C312S, or C391D FLAG-RAD51 were grown to ∼85% confluency. The cells were washed with sterile PBS ( × 3) and incubated in DMEM with 5 mM DAz-2 or 1% (v/v) DMSO for 1.5 h at 37 °C. Afterwards, the cells were washed with cold PBS (×3), scraped into RIPA buffer (250 μl) containing 1x mini complete protease inhibitors (Roche) with a rubber policeman, and lysed by incubating on ice for 15 min with swirling. Lysates were cleared by centrifugation at 4 °C for 15 min (14,000×g) and the protein concentration was determined by BCA assay (Pierce).
3
Phospho-p38 MAPK Western Blotting
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Cells were treated with vehicle (DMSO), Yoda1, or compound 2e at the appropriate times prior to lysing. Cells were harvested in lysis buffer containing 10 mm Tris (pH 7.5), 150 mm NaCl, 0.5 mm EDTA, 0.5% NP-40, MiniComplete protease inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche). A protein quantification assay was then performed using the DC Protein Assay (Bio-Rad). 25 μg of protein was loaded on a 10% polyacrylamide gel. After resolution by electrophoresis, samples were transferred to PVDF membranes, and Western blotting was performed as described previously (38 (link)) using a primary antibody for phospho-p38 MAPK (9215, Cell Signaling Technology). Membranes were reprobed with p38α antibody (9228, Cell Signaling Technology) to confirm equal protein loading. Species-appropriate secondary antibodies (GE Healthcare) and ECL Prime Western blotting detection reagent (GE Healthcare) were used for visualization. Syngene G:BOX Chemi XT4 was used for imaging alongside GeneSys image acquisition software for densitometric analysis.
4
Phosphorylation Analysis Using Phos-tag Gel
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Shisa9/CKAMP44 phosphorylation was assessed using the Phos‐tag system (Wako, Neuss, Germany), which facilitates phosphorylation‐dependent retarded protein migration through a polyacrylamide gel, and subsequent WB. Phos‐tag gel electrophoresis (6–8% polyacrylamide supplemented with 50 μm Phos‐tag) was established using an optimised protocol with Zn2+ in a bis/tris‐buffered neutral pH gel system either as described previously 48 or according to the manufacturer's recommendations. Phos‐tag gels were generally blotted using a wet transfer blotting set‐up. As a control for phosphorylated proteins, the proteins were dephosphorylated using a thermosensitive alkaline phosphatase (FastAP; Thermo Fisher Scientific). The transfected cells were lysed in 1× FastAP buffer (Thermo Fisher Scientific) containing 1% Triton‐X and Mini Complete protease inhibitors without EDTA (Roche). The lysates were cleared by centrifugation and proteins were subsequently dephosphorylated for 2 h at 37 °C using FastAP.
5
Yeast Cell Lysis and Protein Extraction
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S100 Extracts were prepared as previously described25 (link). Briefly, yeast were grown to mid-log phase, and were lysed by beating with glass beads in Lysis Buffer (50 mM Tris pH 7.5, 100 mM KCl, 0.1 mM EDTA, 10 mM MgCl2, 10% glycerol, 10 mM β-mercaptoethanol, 1 mM PMSF, Mini Complete Protease Inhibitors (Roche)). Lysates were clarified by centrifugation, followed by a 20 min spin at 12,000 × g, and a 60 min spin at 100,000 × g.
6
In vitro Deubiquitination of GluA1
7
Western Blot Analysis of Protein Targets
8
Immunoblotting for Protein Expression
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Cells were harvested in lysis buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, Mini Complete protease inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche). 20 µg protein was loaded on a 4–20% gradient gel and resolved by electrophoresis. Samples were transferred to PVDF membranes and labelled overnight with primary antibody: mouse anti-human β-actin (1:2000, Santa Cruz Biotechnology) or mouse anti-human CD31 (1:1000, Abcam). Species appropriate secondary antibodies and SuperSignal Femto detection reagent (Perbio Science) were used for visualisation.
9
Western Blot Analysis of Protein Targets
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Cells were transfected and harvested in lysis buffer containing 10 mM Tris, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.5 % NP-40, MiniComplete protease inhibitors (Roche), and PhosSTOP phosphatase inhibitors (Roche). Equal protein amounts were loaded on 8 % gels and resolved by electrophoresis. Samples were transferred to PVDF membranes and labeled overnight with primary antibody: anti-β-actin (0.2 μg.mL−1, Santa Cruz); anti-total-eNOS (0.2 μg.mL−1, BD Biosciences); anti-p(S1177)-eNOS (0.2 μg.mL−1, BD Biosciences); or anti-Piezo1 (0.3 μg.mL−1, Proteintech). Horse radish peroxidase-conjugated donkey anti-mouse or anti-rabbit secondary antibody (Jackson ImmunoResearch) and SuperSignal Femto detection reagent (Perbio Science) were used for visualization. Densities of protein bands were quantified by ImageJ software. Catalogue information for antibodies: anti-β-actin (0.2 μg.mL−1, Clone C4, sc-47778; Santa Cruz); anti-total-eNOS (0.2 μg.mL−1, Clone 3/eNOS/NOS Type III, Cat. #610297; BD Biosciences); anti-p(S1177)-eNOS (0.2 μg.mL−1, Cat. #612392; BD Biosciences); or anti-Piezo1 (0.3 μg.mL−1, Proteintech).
10
Prostate Cancer Protein Profiling
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Whole cell lysates were prepared by removing the culture media, washing with cold PBS, and lysing cells with 1x RIPA buffer containing Mini-complete protease inhibitors (11836170001, Roche, Basel, Switzerland) and phosphatase inhibitor cocktail (4906845001, Roche). Protein concentrations were determined using the Bradford assay, and 20 μg of protein/well was loaded onto the gels. Standard western blotting procedures were followed, and the following antibodies were used: β-ACTIN (Sigma–Aldrich, A3854), FABP5 (Cell Signaling Technology (Danvers, MA, USA), 39926), AR (Santa Cruz Biotechnology (Dallas, TX, USA), sc-7305), FKBP5 (Cell Signaling Technology, 12210S), NKX3.1 (Santa Cruz Biotechnology, sc-393190), β-Tubulin (Cell Signaling Technology, 2146), and Vinculin (Cell Signaling Technology, 13901). Western blots were imaged using Amersham Hyperfilm ECL (Amersham Bioscience, Buckinghamshire, UK), and the scanned images were analyzed using ImageJ software, v. 1.54f).
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