The RIPA lysis buffer (cat. no. 20-188; Millipore, Burlington, CA, USA) with protease inhibitor cocktail tablets (cat. no. 1836153001; Roche, Basel, Switzerland) and phosphatase inhibitor cocktail tablets (cat. no. 04906837001; Roche, Basel, Switzerland) was used to lyse the cancer cell samples. The same amounts of proteins were separated by molecular weight on 8–12% SDS–polyacrylamide gels. After the separation of the proteins, they were transferred to Amersham™ Protran™ 0.2 µm NC (cat. no. 10600001; Amersham™; Cytiva, Pittsburgh, PA, USA). The transferred membranes were blocked by using phosphate-buffered saline (PBS) buffer containing nonfat milk (10%) for 30 min at room temperature. Membranes were probed with primary antibodies overnight in a 4 °C chamber. The next day, after washing membranes with Tris-buffered saline (TBS) buffer (cat. No. A0027; BIO BASIC, Markham, ON, Canada) containing Tween 20 (0.1%) (cat. No. TB0560; BIO BASIC, Markham, ON, Canada), HRP-linked secondary antibodies (anti-rabbit IgG; cat. No. 7074S or anti-mouse IgG; cat. No. 7076S; Cell Signaling Technology, Inc., Danvers, MA, USA) were treated for 2 h at room temperature. These membranes were visualized by using Western Blotting Luminol Reagent (cat. No. sc-2048; Santa Cruz Biotechnology, Inc., Dallas, TX, USA).
Protease inhibitor cocktail tablet
Manufactured by Merck Group
Sourced in United States, Germany
Protease inhibitor cocktail tablets are a laboratory product manufactured by Merck Group. The core function of this product is to inhibit the activity of proteases, which are enzymes that cleave or break down proteins. These tablets are commonly used in various research and analytical applications to prevent unwanted protein degradation.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Edta free, by Merck Group (3 mentions)
Trypsin, by Promega (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Immobilon p pvdf membrane, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (2 mentions)
Phosphatase inhibitor cocktail 3, by Merck Group (2 mentions)
Amersham ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (2 mentions)
Sodium acetate trihydrate, by Merck Group (2 mentions)
Crystal violet stain, by Merck Group (2 mentions)
Sterile saline, by Henry Schein (2 mentions)
Acetonitrile, by Henry Schein (2 mentions)
Captiva filtration equipment, by Agilent Technologies (2 mentions)
Sheta2, by Cayman Chemical (2 mentions)
Methanol, by Merck Group (2 mentions)
Hydrochloric acid (hcl), by Merck Group (2 mentions)
Phosphoric acid, by Merck Group (2 mentions)
Isoflurane, by Henry Schein (2 mentions)
72 protocols using protease inhibitor cocktail tablet
1
Western Blot Analysis of Cancer Cell Proteins
2
Immunoprecipitation and Immunoblotting Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in an ice‐cold immunoprecipitation buffer (NP‐40 lysis buffer) containing protease inhibitor cocktail tablets (539 131, Millipore) and centrifuged at 13,000 × g for 15 min. The obtained cell lysates were incubated with the indicated antibody on a rocking platform at 4 ℃ overnight. On the second day, cell lysates were incubated with protein A/G‐agarose beads (SC2003; Santa Cruz Biotechnology) for 2 hr at 4℃. The immune complex was collected after washing with cold immunoprecipitation buffer and subjected to immunoblotting with the indicated primary antibodies and corresponding secondary antibodies.
3
Immunoprecipitation of VSV-G Protein Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Doxycycline, monoclonal mouse anti-VSV-G antibody, and anti-VSV-G agarose beads were purchased from Sigma-Aldrich (Gillingham, United Kingdom). Lipofectamine 2000 transfection reagent was purchased from Invitrogen (Paisley, United Kingdom). Protease inhibitor cocktail tablets were purchased from Millipore (Massachusetts, United States). Anti-BAG4, anti-FAR1, anti-PP2Aα, anti-TIM50, anti-AKAP8L, anti-PSMC2, anti-SMC3 and anti-GAPDH antibodies were purchased from Santa Cruz (Heidelberg, Germany). Anti-Raf1, anti-AIF, anti-HAX1, and anti-GRP78 antibodies were purchased from BD Biosciences (Oxford, United Kingdom). The anti-ANT2 antibody was purchased from Cell Signaling Technology (Hitchin, Hertfordshire, United Kingdom). Anti-IRS4, anti-KAP1, anti-RCN1, anti-NDUFA4 and anti-RCN2 antibodies were purchased from Abcam (Cambridge, United Kingdom). Anti-VSV antibody was from sigma Sigma-Aldrich (Gillingham, United Kingdom). Anti-RUVB1 and anti- RUVB 2 antibodies were from Proteintech (Illinois, United States).
4
Quantitative Proteomics of SIRT2 Acetylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibody against GAPDH and SIRT2 was purchased from Abcam. Triton X-100 was bought in Sangon Biotech. Protease Inhibitor Cocktail tablets were purchased from Merck Millipore. Trichostatin A (TSA) was bought in MedChemExpress. Nicotinamide (NAM), trichloroacetic acid (TCA), triethylammonium bicarbonate (TEAB), DTT, iodoacetamide, EDTA, 2-deoxy-glucose (2-DG), TFA, and formic acid (FA) were bought from Sigma-Aldrich. BCA protein quantification kit was bought from Beyotime Biotechnology. Trypsin was purchased from Promega. Anti-acetyl-lysine antibody beads were bought from PTM BIO. Acetonitrile (ACN) was purchased from ThermoFisher Scientific. TRIzol reagent was bought in Invitrogen. FastQuant RT kit was bought from TianGen. Oligomycin was bought from Toronto Research Chemicals.
5
Western Blot Analysis of DNMT3B
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed three times with phosphate buffered solution (PBS) and lysed with lysis buffer (100 mM Tris-HCl (pH8), 140 mM NaCl, 0,2% SDS, 1% NP40, 20 mM EDTA, containing protease inhibitor cocktail tablets (11697498001, MERCK). Protein lysates were quantified by Bradford protein assay (Bio-Rad). 30 μg of total protein lysates were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore), blocked with 2% BSA in TBS buffer with 0.1% Tween20. DNMT3B was detected with anti-DNMT3B diluted 1:3000 (C15410218, Diagenode). For protein loading normalization, membranes were also reacted with an anti-actin antibody diluted 1:5000 (A2066, Sigma). Protein levels were calculated by using Typhoon Scan and ImageQuant 5.2 software.
6
Comprehensive Immunological Assays for In-Depth Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gum arabic, activated carbon, 2,7-dichlorofluorescein diacetate (DCFDA), toluidine blue, protease inhibitor cocktail tablets, phosphatase inhibitor cocktail tablets, MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel were purchased from Merck KGaA (Darmstadt, Germany). 25% Aqueous Solution Glutaraldehyde, Paraformaldehyde, Sorensen's Phosphate Buffer, 2% Aqueous Solution Osmium Tetroxide, Ethyl Alcohol, Acetone, Araldite, Dibutyl phthalate (DBP) were purchased Electron Microscopy Sciences (Hatfield, USA). Leukocyte Activation Cocktail with BD GolgiPlug™, FACS antibodies include anti-mouse I-A/I-E-BV510, IgG1-BB700, IgM-BV605, IgE-BV786, IgD-BV711, CD1d-BV421, CD5-PE, CD45-BUV395, CD19-APC, CD45R/B220-BUV496, CD45-APC-Cy7, CD3e-FITC, CD4-V450, CD8-BV510, CD25-BV605, IL-4-PE-Cy7, IFN-γ-PE, FoxP3-AF647, CD103-BUV395, F4/80-BV711, CD80-BV650, CD11b-BV510, Ly6-G-PerCP Cy5.5, PE-Ly6-C, CD45-APC-Cy7, CD11c--AF700 were purchased from BD (Heidelberg, Germany); Another FACS antibody Anti-mouse-IL-17A-BV650 was purchased from eBioscience (Frankfurt am Main, Germany). Anti-mouse HMGB1, Anti-mouse phospho-p65, anti-mouse phospho-p38, anti-mouse GAPDH and anti-rabbit IgG HRP were obtained from Cell signaling Technology (Frankfurt am Main, Germany). IgE and histamine ELISA kits were purchased from Abcam (Berlin, Germany).
7
Western Blot Analysis of FAN1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed once in DPBS and lysed with RIPA buffer (Sigma-Aldrich) containing cOmplete, EDTA-free Protease Inhibitor Cocktail Tablets (Merck). Protein samples were denatured at 70 °C for 10 minutes in 4× NuPAGE LDS Sample Buffer (Life Technologies). Next, 40 µg of cell protein extract per sample was separated on NuPAGE 4–12% Bis-Tris gradient gels (Life Technologies) alongside PageRuler Plus Prestained Protein Ladder (Thermo Fisher Scientific) using MOPS SDS NuPAGE Running Buffer (Invitrogen) at 200 V for 50 minutes. Where purified proteins were run, 30 ng of protein per lane was loaded. Gels were then transferred to methanol-activated Immobilon-P PVDF membranes (Sigma-Aldrich) using NuPAGE Transfer Buffer (Invitrogen) at 120 V for 45 minutes. The membrane was blocked in 5% milk in PBS-T and incubated overnight at 4 °C with anti-FAN1 (CHDI, sheep polyclonal, 1:1,000) and anti-β-tubulin (UpState, 05-661, mouse monoclonal, 1:10,000). Donkey anti-mouse IgG Alexa Fluor 680 (Invitrogen, A32788, 1:10,000) and IRDye 800CW donkey anti-goat IgG secondary antibody (LI-COR Biosciences, 926-32214, 1:15,000) were used as secondary antibodies. Immunoblots were visualized with the Odyssey CLx Imaging System using β-tubulin as a loading control.
8
Cell Lysis and Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were rinsed twice with ice-cold PBS and lysed with RIPA lysis buffer (20 mM Tris pH 7.4, 137 mM NaCl, 10% glycerol, 1% Triton X-100, 0.5% deoxycholate, 0.1% SDS, 2.0 mM ethylenediaminetetraacetic acid (EDTA, pH 8.0), complete EDTA free Protease inhibitor cocktail tablets (1 tablet per 50 mL RIPA) (Sigma-Aldrich Israel, Rehvot, Israel, #11836170001) and phosphatase inhibitor cocktail (100×) (Bimake, Houston, TX, USA, # BI5001-A and B). The lysates were cleared by centrifugation at 13,000 rpm at 4 °C in a microcentrifuge for 10 min. The protein concentration was determined by Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA, #5000006). Proteins were denatured by adding SDS sample buffer (5×) and boiling for 5 min, then resolved by 10% SDS-PAGE, transferred onto a 0.45 µm PVDF membrane (Immobilon-P Transfer membrane) (Merck Millipore, Carrigtwohill, Ireland, Co, #IPV00010), and probed with the appropriate antibodies.
9
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA buffer (150 mM NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 10 mM Tris-HCl pH 7.2, and 5 mM EDTA), supplemented with phosphatase inhibitors and protease inhibitor cocktail tablets (Sigma-Aldrich, St. Louis, MO). Protein concentration was measured by BCA assay (Thermo Scientific-Pierce, Rockford, USA). Protein samples were subjected to SDS-PAGE and blotted to Immobilon-P PVDF membranes (Millipore Co., Massachusetts, USA). Membranes were blocked in PBS-tween 0.1% with 5% nonfat dried milk for 1 h at 25°C and then incubated with primary antibodies overnight at 4°C. After extensive washing with PBS-tween 0.1%, membranes were incubated with peroxidase-conjugated secondary antibodies. Signal was developed by chemiluminescence (ECL, Amersham Pharmacia Biotech, Little Chalfont, UK) and acquired on a ChemiDocTR XRS+ high definition system (Bio-Rad). Bands corresponding to the different proteins were digitalized employing the Image Lab version 3.0.1 (Bio-Rad Laboratories). This assay was performed at least three times for each target.
10
Investigating P-glycoprotein Regulation by D-ME and SD-ME
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
EC109 and EC109/DDR cells were treated with various concentrations of D-ME and SD-ME for 48 h, and then the levels of P-gp and β-actin were measured by western blotting. Total protein from the cells was extracted in RIPA lysis buffer (Beyotime, Nanjing, China) supplemented with protease inhibitor cocktail tablets (Sigma, USA) and quantified using a BCA protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA). A total of 30 μg of protein was separated using 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a nitrocellulose (NC) membrane (Millipore, MA). The membrane was blocked with 5% nonfat milk and incubated at 4 °C overnight with the desired primary antibody. After washing three times with phosphate buffer solution and Tween 20 (PBST), the membrane was incubated for 60 min with horseradish peroxidase-conjugated secondary antibody diluted in PBST. Protein bands were visualized using enhanced chemiluminescence (Millipore, USA) and detected using a Bio Imaging System (Clinx, China). The relative protein levels were normalized to β-actin as a loading control.
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!