Cells or organoids were lysed using RIPA buffer containing a complete mini protease inhibitor cocktail (Roche) and PhosphoSTOP phosphatase inhibitor cocktail (Roche). Protein was quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher). Western blot analysis was performed using 30 μg of protein lysates, as described in [5 ]. The antibodies used are described in Table S1 . We thank Prof. John Silke (WEHI) for providing the c-IAP2 antibody as a gift.
Phosphostop phosphatase inhibitor cocktail
Manufactured by Roche
Sourced in Switzerland, Germany
PhosphoStop Phosphatase Inhibitor Cocktail is a laboratory reagent designed to inhibit the activity of phosphatases. It is used to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Complete mini protease inhibitor cocktail, by Roche (4 mentions)
Complete edta free protease inhibitor cocktail, by Roche (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc xrs system, by Bio-Rad (2 mentions)
Hypodermic needle pro, by B. Braun (2 mentions)
Mini wet tank blotting system, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Image lab 5, by Bio-Rad (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Image studio 4, by LI COR (1 mentions)
Odyssey clx, by LI COR (1 mentions)
Pierce 660 nm protein assay kit, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Anti ripk1 antibody, by Cell Signaling Technology (1 mentions)
Ab108217, by Abcam (1 mentions)
Protein a agarose beads, by Cell Signaling Technology (1 mentions)
Mouse anti cleaved caspase 8, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
10 protocols using phosphostop phosphatase inhibitor cocktail
1
Western Blot Analysis of Protein Lysates
2
Protein Extraction and Analysis from Embryos
3
Toxin-induced cell morphology changes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
At a confluency of 75% medium was replaced with 20 mL of fresh medium. For treatment 255 ng/mL of TcnA or FA_TcnA were added to 20 mL medium. Control cells were grown in medium without addition of toxin. Morphological changes of cells were documented by phase-contrast microscopy after 24 h. These conditions were chosen in order to reach maximal percentage of rounded cells without any undesired side effects as necrosis. Cells were washed twice with ice-cold PBS and stored at −80 °C overnight. The cells were then lysed by scraping them in 300 μL lysis buffer (8 M Urea, 50 mM ammonium bicarbonate (pH 8.0), 1 mM sodium ortho-vanadate, complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany), and phosphoSTOP phosphatase inhibitor cocktail (Roche, Mannheim, Germany)). Lysates were sonicated on ice for 15 s at 30% energy output. Cell debris was removed by centrifugation for 15 min at 13,000× g at 4 °C. The supernatant was used directly or stored at −80 °C.
4
Cellular Effects of Cytolethal Distending Toxin
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Two days before toxin treatment, 7.5 × 105 cells were seeded in 10 cm dishes to achieve a 75% confluency on the day of toxin treatment. CDTa and CDTb were generated using an Escherichia coli expression system as previously described (Beer et al., 2018 ). Cells were treated with 1.5 μg/mL CDTa and 3 μg/mL CDTb. Changes in the morphology were documented by phase-contrast microscopy after 4 and 8 h, respectively. For controls, only medium was exchanged. While LPS contamination was not separately checked by an assay, classical LPS reaction pathways, e.g., ERK pathway, show no activation (Supplementary Tables 1 , 2 ), and no inflammatory GO terms were enriched in GO analysis indicating that there is no major additional effect concerning LPS. After documentation, cells were washed twice with ice-cold PBS and lysed by scraping them into 600 μL lysis buffer [8 M Urea, 50 mM ammonium bicarbonate (pH 8.0), 1 mM sodium ortho-vanadate, complete EDTA-free protease inhibitor cocktail (Roche), and phosphoSTOP phosphatase inhibitor cocktail (Roche)]. Lysates were sonicated on ice two times for 5 s at 30% energy. Cell debris was removed by centrifugation for 15 min at 16,100 × g at 4°C. The supernatant was collected and used for further preparation.
5
Immunoprecipitation of Complex-I, RIPK1, and SMYD2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation of complex-I, cells were seeded in 15 cm Petri dishes and treated with 1 µg/ml FLAG-TNF (ALX-522-009-C050 for MC-38 cells and ALX-522-008-C050 for HT-29 cells; Enzo Life Sciences). Whole-cell extracts were prepared in the presence of native lysis buffer (9803; Cell Signaling Technology) supplemented with protease inhibitors (Complete Mini Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosphoStop Phosphatase Inhibitor Cocktail, Roche). For the control samples, 1 µg/ml FLAG-TNF was added post-lysis. Cell lysates were centrifuged for 20 min at 14,000 rpm and supernatants were collected and incubated with Anti-FLAG® M2 Magnetic Beads (M8823; Sigma-Aldrich) overnight at 4 °C. The beads were washed five times with TBS and co-precipitated proteins were eluted using SDS–PAGE loading buffer.
For immunoprecipitation of RIPK1 and SMYD2, cell lysates were prepared as mentioned above but incubated with anti-RIPK1 antibody (3493; Cell Signaling Technology) or anti-SMYD2 antibody (ab108217; Abcam) overnight at 4 °C. The next day, Protein A Agarose Beads (9863; Cell Signaling Technology) were added into the mixture and incubated in room temperature for 30 min. The beads were washed five times with native lysis buffer and co-precipitated proteins were eluted using SDS–PAGE loading buffer.
For immunoprecipitation of RIPK1 and SMYD2, cell lysates were prepared as mentioned above but incubated with anti-RIPK1 antibody (3493; Cell Signaling Technology) or anti-SMYD2 antibody (ab108217; Abcam) overnight at 4 °C. The next day, Protein A Agarose Beads (9863; Cell Signaling Technology) were added into the mixture and incubated in room temperature for 30 min. The beads were washed five times with native lysis buffer and co-precipitated proteins were eluted using SDS–PAGE loading buffer.
6
Immunoprecipitation of Flag-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation, whole-cell extracts were prepared in the presence of native lysis buffer [20 mM Tris-HCl (PH 7.5), 100 mM NaCl, 10% glycerol, 0.5% NP-40, and 0.5% DDM] supplemented with protease inhibitors (Complete Mini Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (PhosphoStop Phosphatase Inhibitor Cocktail, Roche). Cell lysates were centrifuged for 20 min at 19,000 g and supernatants were collected and incubated with anti-Flag (M2) agarose for 1 h at room temperature. The agarose beads were washed five times with native lysis buffer and co-precipitated proteins were eluted using SDS–PAGE loading buffer.
7
Protein Isolation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were isolated from cells or tissues using Mammalian Protein Extraction reagent (Thermo Fisher Scientific) supplemented with protease inhibitors (Complete Mini Protease Inhibitor Cocktail; Roche) and phosphatase inhibitors (PhosphoStop Phosphatase Inhibitor Cocktail; Roche). Proteins were separated using a MiniProtean-TGX gel (4–15% polyacrylamide; Bio-Rad Laboratories) and transferred from the gel to a nitrocellulose membrane (Whatman). Membranes were probed with the following primary antibodies: anti–mouse RIP3 (ADI-905-242-100; Enzo Life Sciences), anti–human RIP3 (13526; Cell Signaling Technology), anti–mouse cleaved caspase-8 (8592; Cell Signaling Technology), anti–caspase-8 (4790; Cell Signaling Technology), anti–human phospho-MLKL (S358, ab187091; Abcam), and HRP-linked β-actin (ab49900; Abcam). HRP-linked anti–rabbit IgG (7074; Cell Signaling Technology) was used as a secondary antibody.
8
Immunoblotting Analysis of ALK Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in buffer containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Triton X-100, 5 mM EDTA, 1 mM Na3VO4, proteinase inhibitors cocktail (Roche) and PhosphoStop phosphatase inhibitor cocktail (Roche). Proteins were separated by electrophoresis on 4-20% polyacrylamide gradient gels, transferred onto membranes using iBlot Transfer Stack (nitrocellulose membrane) with iBlot Transfer System (Invitrogen). Proteins were detected by immunoblotting using an enhanced chemiluminescence system (Perkin-Elmer). Anti-ALK, anti-phospho-ALK (Tyr1604), anti-phospho-ALK (Tyr1096), anti-phospho-ALK (Tyr1078), anti-phospho-CRKL (Tyr207), anti-CRKL, anti-ERK1/2 and anti-phospho-ERK1/2 antibodies were obtained from Cell Signaling Technology. The anti-α-tubulin antibody was purchased from Sigma-Aldrich.
9
Whole-Cell Lysate Preparation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared in RIPA lysis buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate) supplemented with 1 mM PMSF, 1% SDS, and phosphatase inhibitors (1x PhosphoSTOP Phosphatase Inhibitor Cocktail, Roche Diagnostics, Cat#4906845001). Samples were lysed on ice for 20 min, sheared by passing cells through a G25 needle (B. Braun, Hypodermic Needle-Pro®, Cat#4658304), cleared by centrifugation (14,000 × g, 15 min), and quantified using Pierce™ Bradford protein assay kit (Thermo Scientific, Cat#23200). Lysates were boiled 5 min at 95 °C with 6x reducing Laemmli buffer (0.12 M Tris pH 6.8, 47% glycerol, 12% SDS, 0.6 M DTT, 0.06% bromophenol blue). Samples were separated on a polyacrylamide gel and transferred to PVDF membranes using the mini wet/tank blotting system (Bio-Rad). After blocking with 5% BSA in Tween-20/PBS, membranes were probed with primary antibodies prepared in blocking solution overnight at 4 °C on a roller, followed by incubation with horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 h at room temperature and ECL detection (Thermo Fisher, Cat#34096) by the ChemiDoc XRS + system (Bio-Rad). Primary and secondary antibodies used for immunoblotting are listed in Additional file 8 : Table S1. Quantitative analysis of protein expression relative to GAPDH was done using Image Lab software (Bio-Rad).
10
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell lysates were prepared in RIPA lysis buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 0.1% SDS, 0.5% Sodium deoxycholate) supplemented with 1mM PMSF, 1% SDS, and phosphatase inhibitors (1x PhosphoSTOP Phosphatase Inhibitor Cocktail, Roche Diagnostics, Cat#4906845001). Samples were lysed on ice for 20 min, sheared by passing cells through a G25 needle (B. Braun, Hypodermic Needle-Pro®, Cat#4658304), cleared by centrifugation (14000 x g, 15 min), and quanti ed using Pierce TM Bradford protein assay kit (Thermo Scienti c, Cat#23200). Lysates were boiled 5 min at 95° C with 6x reducing Laemmli buffer (0.12M Tris pH 6.8, 47% glycerol, 12% SDS, 0.6M DTT, 0.06% bromophenol blue). Samples were separated on a polyacrylamide gel and transferred to PVDF membranes using the mini wet/tank blotting system (Bio-Rad). After blocking with 5% BSA in Tween-20/PBS, membranes were probed with primary antibodies prepared in blocking solution overnight at 4° C on a roller, followed by incubation with horseradish peroxidase-conjugated secondary antibody in blocking solution for 1 h at room temperature and ECL detection (Thermo Fisher, Cat#34096) by the ChemiDoc XRS+ system (Bio-Rad). Primary and secondary antibodies used for Western blotting are listed in Supplementary Table S1. Quantitative analysis of protein expression relative to GAPDH was done using Image Lab software (Bio-Rad).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!