Protein lysates were obtained using radioimmunoprecipitation assay (RIPA) buffer supplemented with the protease and phosphatase inhibitor cocktail (Bimake). Grinding and ultrasonication were additionally used for protein extraction from the clinical specimens and murine mandibles. Protein was quantified using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific). Extracts of 20 μg of total protein were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred onto the polyvinylidene fluoride membranes (Bio-Rad). After being blocked, the membranes were incubated with specific primary antibodies for RIPK3 (1:500, Santa Cruz Biotechnology; 1:1,000, Novus Biologicals), p-RIPK3 (1:1,000, Abcam; 1:1,000, Cell Signaling Technology), MLKL (1:1,000, Abcam; 1:1,000, Cell Signaling Technology), p-MLKL (1:1,000, Abcam; 1:1,000, Cell Signaling Technology), and β-actin (1:5,000, Abclonal) at 4°C overnight. The membranes were then incubated with the secondary antibodies conjugated with horseradish peroxidase (1:5,000, Proteintech). Afterward, the membranes were visualized using bio-Femto enhanced chemiluminescence (Fudebio) and a chemiluminescent imaging system (Tanon 5200). ImageJ software was used for the analysis of optical density of the immunoblot bands.
Protease and phosphatase inhibitor cocktail
Manufactured by Selleck Chemicals
Sourced in United States, China
Protease and phosphatase inhibitor cocktails are laboratory reagents designed to inhibit the activity of proteases and phosphatases in biological samples. These cocktails contain a mixture of chemical compounds that work together to prevent the degradation of proteins and the dephosphorylation of phosphorylated biomolecules during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Beyotime (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Bca assay, by Thermo Fisher Scientific (3 mentions)
Phgdh, by Merck Group (3 mentions)
Tris glycine gel, by Thermo Fisher Scientific (3 mentions)
Mg132, by Selleck Chemicals (3 mentions)
Chemidoc mp imaging system, by Bio-Rad (3 mentions)
Image studio lite, by LI COR (3 mentions)
Rhodamine conjugated anti tubulin, by Bio-Rad (2 mentions)
Ripa buffer, by Boston BioProducts (2 mentions)
Cell recovery solution, by Corning (2 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (2 mentions)
Protease and phosphatase inhibitor, by Roche (2 mentions)
Ecl detection reagent, by Beyotime (2 mentions)
Protein g agarose, by Thermo Fisher Scientific (2 mentions)
Amersham imager 600, by GE Healthcare (2 mentions)
Ripa lysis buffer, by Meilun (2 mentions)
Stat3, by Cell Signaling Technology (2 mentions)
35 protocols using protease and phosphatase inhibitor cocktail
1
Western Blot Analysis of Cell Death Proteins
2
Protein Extraction and Western Blot Analysis
3
Western Blot Analysis of Protein-Protein Interactions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (50 mM Tris-HCl [pH 8.0], 5 mM EDTA, 150 mM NaCl, and 0.5% Nonidet P-40 and a protease and phosphatase inhibitor cocktail (Bimake, China)), and the clarified lysates were resolved by SDS-PAGE and transferred to PVDF membranes for Western blot using ECL detection reagents (Beyotime, China). The immunoblots were processed according to standard procedures using primary antibodies directed to FBW7 (Bethyl, A301-720A), BRD7 (Bethyl, A302-304A), Cyclin E (CST, 20808), GAPDH (CST, 2118), HA (CST, 3724), Flag (CST, 14793), Myc (CST, 2278), V5 (CST, 13202), RCC2 (CST, 5104), RAD51 (CST, 8875), ERα (Santa Cruz, SC-514857), tubulin (Santa Cruz, SC-166729), NFATc1 (Abiocode, R2315–1). For immunoprecipitation, the supernatants were first incubated with S-protein agarose (Novagen) overnight at 4 °C, and the precipitates were washed three times with NETN buffer. To detect endogenous interaction, the clarified supernatants were first incubated with anti-FBW7 or NFATc1 antibody for two h and then protein G-agaroses (Thermo Fisher, 10004D) overnight. After washed three times with NETN buffer, the samples were collected and analyzed by Western blot.
4
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in mammalian cell lysis buffer (50mM Tris pH 7.5, 150mM NaCl and 0.5% NP40) containing a protease and phosphatase inhibitor cocktail (Bimake.com ) and 1 μM MG132 (Selleckchem). To generate PDO protein lysates, organoids were incubated for 1 h on ice in Cell Recovery solution (Corning, 354253) and lysed using RIPA buffer (Boston Bioproducts) supplemented with protease and phosphatase inhibitors (Roche). Protein concentration was determined by BCA assay (Thermo Fisher). Quantified protein samples were separated by electrophoresis on 4–20% ready-made Tris-Glycine gels (Invitrogen) and transferred to PVDF membranes (Millipore). Membranes were blocked with 2% bovine serum albumin for 1 h and incubated overnight with one or more primary antibodies: PSAT1 (Thermo Fisher, PA5-22124.), PHGDH (Sigma, HPA021241), PSPH (Santa Cruz, sc-365183), ERα (Cell Signaling, 8644) and Actin (Sigma, A1978). Overexpression and knockout of PSAT1 (Figure 3 ) confirmed the correct band on PSAT1 western blots. Membranes were washed with tween 20-containing tris buffered saline and incubated with fluorescence- or HRP-conjugated secondary antibodies (Bio-Rad). Rhodamine-conjugated anti-tubulin was also treated as a secondary antibody (Bio-Rad, 12004166). Images were detected using a ChemiDoc MP Imaging System (Bio-Rad).
5
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were harvested and total protein was extracted using RIPA buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM EDTA supplemented with a protease and phosphatase inhibitor cocktail (Bimake, Shanghai, China). Western blotting was performed as previously described [50 (link)]. Primary antibodies against hPD-1, hPD-L1, and GAPDH were purchased from Abcam Inc. (Cambridge, MA, USA).
6
Immunoprecipitation and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total cells were lysed in IP and western blot buffer (P0013, Beyotime Biotechnology) containing protease and phosphatase inhibitor cocktail (B14001 and B15001, Bimake). Whole lysates were incubated with 10 μg of antibodies or normal IgG. Briefly, bound proteins were eluted with 0.1 M glycine (pH 2.5) and then neutralized with 1 M Tris buffer to prevent disturbance of the heavy chain (approximately 55 kDa). The elution was analyzed by western blot.
7
Skeletal Muscle Protein Abundance Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The methods employed for Western blotting have been provided in detail previously [22 (link)]. Briefly, skeletal muscle tissue (~40 mg) was stabilized in buffer with protease and phosphatase inhibitor cocktail (Bimake, USA) and homogenized twice for 4 min (10,000 Bullet Blender 24 Gold, Next Advance, USA). Protein content of lysate were quantified (Pierce BCA Protein Assay Kit, Thermo Scientific, USA) samples were equilibrated in 4 × Laemmli sample buffer with DTT and heated to 95°C for 10-min with 50 μg protein subsequently loaded into separate wells on acrylamide gels (Bio-Rad, USA) for electrophoresis and wet-transferred (120-min, 70 V; Polyvinylidene difluoride membrane, Bio-Rad, USA). Membranes were washed (3 × 5-min) in Tris-buffered saline with Tween (0.05%, TBST), then blocked at room temperature for 60 min in TBST and 5% skim milk powder. Membranes were incubated in puromycin primary antibody at 4°C overnight (1: 1000 MABE343, Merck Millipore, MA, USA) and then washed and incubated in secondary antibody. Chemiluminescent solution (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific Inc., USA) was used to quantify blots by densitometry (ChemiDoc, Bio-Rad, USA) and quantified relative to total protein abundance (Amido Black Staining Solution 2 ×, Sigma-Aldrich, USA).
8
Comprehensive Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins from cells and tissues were extracted with RIPA lysis buffer (Meilunbio, Dalian, China) containing protease and phosphatase inhibitor cocktail (Bimake, Beijing, China). The concentration of protein was measured using BCA Protein Assay Kit (Generay, Shanghai, China). Equal amounts of proteins (20 μg) were separated by 8% to 15% SDS-PAGE, then transferred to a PVDF membrane (Bio-Rad, Hercules, CA). The membrane was blocked with 5% non-fat milk in Tris-buffered saline plus Tween 20 (1‰; Meilunbio, Dalian, China) solution, and incubated with the primary antibody overnight at 4°C, followed by incubation with appropriate horseradish peroxidase-conjugated secondary antibodies (Bioker, Hangzhou, China) for 1 h at room temperature. Signals were visualized using enhanced chemiluminescence (Amersham Imager 600; GE, Boston, MA). The antibodies obtained from different companies were as follows: Cell Signaling Technology (ATG7, 8558; ATG5, 12994; ATG12, 4180; Beclin-1, 3495; β-actin, 4970; Bax, 2772; Cleaved caspase-3, 9664; Bcl-xL, 2764; γH2AX, 9718; E-cadherin, 3195; p-STAT3, 9145; STAT3, 9139; ERK, 9102; p-ERK, 4370; cyclinD1, 2978), LC3 (Sigma, L7543), p62 (R&D, MAB8028), Thermo (ZO-1,61–7300; Occludin, 71–1500), Huabio (β-tubulin, 0807-2; GAPDH, EM1101).
9
Immunoprecipitation of Polyubiquitinated NFATc1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
This procedure was performed as previously described [54 (link)]. Briefly, HeLa cells were transfected with the indicated plasmids for 24 h and were treated with 10 μM MG132 for 6 h prior to harvesting. The cells were lysed in RIPA buffer with protease and phosphatase inhibitor cocktail (Bimake, China). Endogenous NFATc1 was immunoprecipitated using anti-NFATc1 antibody for 12 h at 4 °C. Polyubiquitinated NFATc1 was detected using an anti-HA antibody.
10
Immunoblotting Cell Lysate Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysate for immunoblotting was performed according to the standard protocol. Briefly, cells were lysed with buffer that freshly added protease and phosphatase inhibitor cocktail (B15001, Bimake); then, 4%–20% SDS polyacrylamide gels was used to separate proteins, following transferred onto 0.22-μm NC membranes (Thermo Fisher Scientific), and incubated with primary antibodies at 4 °C overnight. The next day, HRP-conjugated secondary antibodies were incubated at room temperature (RT) for 2 h. β-actin was used for the total protein loading control. The primary antibodies and dilutions as follows: GPR4(1:1000, ab75330, Abcam), Phospho-MST1 (T183) (1:1000, 49,322, Cell signalling), MST1(1:1000, 3682, Cell signalling), Phospho-LATS1/2(T1079 + T1041)(1:1000, ab111344, Abcam), Phospho-YAP1 (S127) (1:1000, 13,088, Cell signalling), YAP1(1:1000, 14,074, Cell signalling), β-actin (1:3000, ab8226, Abcam).
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!