Postmortem MFG tissues were homogenized in TBS buffer at a ratio of 1:10 (wt/vol) with Pierce Protease and Phosphatase Inhibitor Cocktail (A32965, ThermoScientific) on ice. Tissue lysate was sonicated and then centrifuged at maximum speed for 15 min at 4 °C. Protein concentrations were measured using the BCA protein assay kit (Bio-Rad Laboratories, Inc.). Electrophoresis was performed using 30 μg of protein lysates, resolved in a 4–12% SDS-PAGE gel (CriterionTM TGXTM, Bio-Rad Laboratories, Inc.) and transferred to a nitrocellulose membrane (Immobilon®-P, Millipore) that was blocked with 5% BSA in TBS with 0.01% tween, followed by overnight incubation of primary antibodies; HT7 (1:300, MN1000, Thermo Fisher), AT8 (1:1000, MN1020, Invitrogen) and PHF1 (1:1000, Peter Davies antibodies) diluted in the blocking solution. Horseradish peroxidase (HRP) secondary antibodies (goat anti-mouse HRP conjugated (1:10,000, 626820, Invitrogen) were incubated for 2 h at RT and the proteins were detected with Supersignal West Pico (34580, Thermo Scientific) and imaged by using iBright 1500 (Invitrogen). Western blots were analyzed using ImageJ Fiji.
Pierce protease and phosphatase inhibitor cocktail
Manufactured by Thermo Fisher Scientific
The Pierce Protease and Phosphatase Inhibitor Cocktail is a ready-to-use solution that inhibits a broad spectrum of proteases and phosphatases. It is designed for use in sample preparation to prevent protein degradation and dephosphorylation.
Automatically generated - may contain errors
Lab products found in correlation
Mn1020, by Thermo Fisher Scientific (1 mentions)
Criterion tgxtm, by Bio-Rad (1 mentions)
Mn1000, by Thermo Fisher Scientific (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Ibright 1500, by Thermo Fisher Scientific (1 mentions)
Omni tip homogenizer, by Omni International (1 mentions)
Cleaved casp3, by Cell Signaling Technology (1 mentions)
Raf 1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Cleaved parp, by Cell Signaling Technology (1 mentions)
Phospho akt, by Cell Signaling Technology (1 mentions)
Phospho s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
S6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Phospho mek1 2, by Cell Signaling Technology (1 mentions)
T per tissue protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
7 protocols using pierce protease and phosphatase inhibitor cocktail
1
Quantitative Proteomic Analysis of Alzheimer's Disease
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare whole-cell lysates, cells were washed twice with cold PBS and lysed in M-PER Mammalian Protein Extraction Reagent (product no. 78501, Thermo Fisher Scientific) supplemented with Pierce protease and phosphatase inhibitor cocktail (product no. A32961, Thermo Fisher Scientific). Tumor tissue samples were lysed in T-PER Tissue Protein Extraction Reagent (product no. 78510, Thermo Fisher Scientific) with above supplement. The automatic hand-operated OMNI-TIP Homogenizer (Omni International, Inc.) was used to homogenize the tumor tissues. Lysates from whole cells and tumor homogenates were then processed for SDS-PAGE Western blotting. The following antibodies were used: Phospho-MEK1/2 (S217/221; catalog no. 9154S), MEK1/2 (catalog no. 4694S), Phospho-AKT (S473; catalog no. 9271S), Phospho-AKT (T308; catalog no. 4056S), AKT (catalog no. 9272S), Phospho-Erk1/2 (catalog no. 4370S), Erk1/2 (catalog no. 4695S), Phospho-S6 Ribosomal Protein (Ser 235/236; catalog no. 4858S), S6 Ribosomal Protein (catalog no.2217S), cleaved-CASP-3 (catalog no. 9664), cleaved-PARP (catalog no. 9541) from Cell Signaling Technology; anti-β-ACTIN (catalog no A5441-.2ML from Sigma-Aldrich; KRAS (catalog no. OP24, from EMDMillipore), and RAF1 (catalog no. sc-7267, Santa Cruz Biotechnology), horseradish peroxidase–conjugated goat anti-GST antibody (catalog no. A190-121P from Bethyl Laboratories).
3
Immunoblotting of Akt and Foxo1 Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD4+ T cells were grown in Treg-inducing conditions with either B7x-Ig or Control-Ig stimulation as described above in 6-well cell culture dishes. After 24 h, the medium was aspirated and the cells were lysed with RIPA buffer (1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris, 150 mM NaCl) supplemented with Pierce protease and phosphatase inhibitor cocktail (Thermofisher). Total protein in the lysates were measured by BCA protein assay (Thermofisher). Equal total protein volumes were run on ExpressPlus 4–12% gels (Genscript) and wet-transferred to nitrocellulose membranes (Thermofisher). EZ-Run Prestained protein ladder (Fisher Scientific) was used for monitoring transfer efficiency and approximating molecular weights. The membranes were blocked with 5% milk in TBST for 1 h at room temperature, then probed with primary antibodies anti-Akt, anti-p-Akt, anti-Foxo1, anti-p-Foxo1 (CST) overnight at 4 °C, and then stained with secondary anti-rabbit HRP (CST) for 1 h at room temperature. Blots were detected with Pierce ECL substrate kit (Thermofisher) and visualized with the ChemiDoc imaging system (Bio-Rad).
4
Immunoprecipitation Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoprecipitation assays, cells were lysed using non-denaturing lysis buffer (25 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% sodium deoxycholate, 4 mM EDTA, 5 mM β-glycerophosphate, 50 mM NaF, 10 mM sodium pervanadate, Pierce protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). 500 µg of total protein lysate was incubated with 5 µg of either rabbit anti-GPRC5A (#HPA007928, Sigma Aldrich), or non-immune rabbit IgG, or 1µg of Anti-Phosphotyrosine Antibody, clone 4G10 (#05-321, EMD Millipore) for 3 h at 4°C, followed by precipitation using 100 µl of SureBeads protein G magnetic beads (BioRad) at 4°C overnight. Beads-bound immunocomplexes were washed 3 times with TBS-0.05% tween. Bound proteins were then eluted using Glycine-HCl buffer, pH 2.0 and analyzed by immunoblotting as described above.
5
Western Blot Analysis of PMS1 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RPE1-AAVS1-CAG115 cells grown to confluency in 6-well plates were rinsed once with ice-cold PBS and lysed in situ with 100 μl per well of ice-cold RIPA buffer (Boston BioProducts, BP-115) containing Pierce™ protease and phosphatase inhibitor cocktail (Thermo Scientific, A32959). Lysates were incubated on a rotary mixer for 30 min at 4 °C followed by centrifugation at 16000 g for 10 min (4 °C) and collection of the supernatant. Protein concentration was measured in the extracts with Pierce™ BCA assay kit (Thermo Scientific, 23225). Protein extracts (20 μg/lane) were resolved on NuPage™ 10% Bis-Tris (Invitrogen, NP0303) or 3–8% Tris-acetate mini gel (Invitrogen, EA03755) and transferred to 0.45 μm nitrocellulose membrane (Thermo Scientific, 88018). The membrane was blocked for 1 h at RT in 5% non-fat dried milk in TBST (Tris-buffered saline with 0.1% Tween 20). Primary antibodies: PMS1 (mouse monoclonal 68413-1-Ig, Proteintech, 1:4000), β-actin (rabbit polyclonal 4967 S, Cell Signaling Technology, 1:1000), in blocking solution, were applied overnight at 4 °C. Secondary antibodies were horseradish peroxidase-linked anti-mouse and anti-rabbit IgG (NA931 and NA934, respectively; GE Healthcare), both 1:4000 in blocking solution, incubated for 2 h at RT. Western blots were developed with Pierce™ ECL kit (Thermo Scientific, 32106).
6
THOP1 Protein Quantification in Cells and Secretome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cellular or extracellular/secreted protein levels of THOP1 were determined on total protein extracts and conditioned media (20× concentrated), respectively, using The RayBio Human Thimet Oligopeptidase ELISA Kit (ELH-THOP1; Raybiotech). To concentrate secreted proteins in cell-conditioned media, serumfree medium was collected after being conditioned overnight and concentrated using Amicon ultra-15 Centrifugal Filter Units with 50 kDa cut-off (30 minutes, 4,000 × g, 4°C). Following centrifugation, supernatant was diluted to reach a final concentration factor of 20 (starting volume/assay volume) and was analyzed for THOP1 protein levels. In parallel, cells were placed on ice and intracellular proteins were extracted in RIPA buffer with Pierce protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). Ten micrograms of total proteins was used for quantification of cellular THOP1 protein expression.
7
Cytokine and IgE Quantification in Lung Homogenate
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole lungs were homogenized in 1 ml PBS containing 0.05% Triton X-100, Pierce Protease and Phosphatase Inhibitor cocktail (Thermo Scientific™). Suspensions were filtered with a 40 μm cell strainer and clarified by centrifugation. Supernatant was used for detecting cytokines by ELISA [30 (link)]. The levels of IL-4, IL-5, IL-13, IL-33, and IgE were measured with sandwich ELISA kit according to the manufacturer’s instructions. For IL-33 in lung homogenate, also total protein determination was performed from the same sample, and the results were then presented in relation to total protein concentration for each sample. LDH release was detected in BALF and cells supernatant using the LDH Cytotoxicity Assay Kit (88953; Thermo Scientific) according to the manufacturer’s instructions. Readings were carried out at a 490 nm wavelength, using a microplate reader (Thermo Scientific) and expressed as % LDH release.
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!