Frozen brain tissues were lysed using cell lysis buffer (Cell Signaling Technology, MA, United States) supplemented with 400 μM PMSF protease inhibitor (Cell Signaling Technology, MA, United States) and incubated on ice for 30 min. Lysates were subjected to centrifugation at 12,000 × g for 30 min at 4°C and protein concentrations were determined by Pierce BCA assay (Thermo Fisher Scientific, MA, United States). A total of 50 μg of protein lysates were boiled for 10 min and subjected to SDS-PAGE electrophoresis using 4–15% precast gels (Bio-Rad). Densitometry was calculated using the Image Lab Software 5.2.1 (Bio-Rad Laboratories, CA, United States). Antibodies used in this study are as follows: anti-Frataxin (1:250) (Abcam Cat# ab113691, RRID:AB_10862125 ), anti-Tubulin (1:10,000) (Abcam Cat# ab6160, RRID:AB_305328 ), rabbit anti-Rat HRP (1:5000) (Abcam Cat# ab6734, RRID:AB_955450 ), and goat anti-Mouse HRP (1:300) (Agilent Cat# P0447, RRID:AB_2617137 ). Non-relevant gel lanes and unspecific bands were excised by digital treatment using Power Point. Original uncropped images are presented as Supplementary material .
Pmsf protease inhibitor
Manufactured by Cell Signaling Technology
Sourced in United States
PMSF (phenylmethylsulfonyl fluoride) is a serine protease inhibitor. It irreversibly inhibits the activity of serine proteases, such as trypsin, chymotrypsin, and thrombin, by binding to the active site of the enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software 5, by Bio-Rad (3 mentions)
Cell lysis buffer, by Cell Signaling Technology (3 mentions)
Ab113691, by Abcam (2 mentions)
Bradford assay, by Bio-Rad (2 mentions)
Precast gel, by Bio-Rad (2 mentions)
P0447, by Agilent Technologies (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Ab6160, by Abcam (1 mentions)
Pierce bca assay, by Thermo Fisher Scientific (1 mentions)
A2066, by Merck Group (1 mentions)
Bca protein assay reagent, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit hrp, by Agilent Technologies (1 mentions)
Goat anti mouse hrp, by Agilent Technologies (1 mentions)
Anti frataxin, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Pierce ecl plus, by Thermo Fisher Scientific (1 mentions)
Image lab software version 5, by Bio-Rad (1 mentions)
Chemidoc system, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Nitrocellulose, by GE Healthcare (1 mentions)
6 protocols using pmsf protease inhibitor
1
Frataxin Protein Expression Analysis
2
Frataxin Expression Modulation by Epigenetic Inhibitors
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Cells were treated with 100 nM BIX01294, 2 μM GSK126 or combination of both for 72 h. Cells were washed with ice-cold PBS and lysed on ice for 30 min with cell lysis buffer (Cell Signaling Technology, 9803) supplemented with 400 μM PMSF protease inhibitor (Cell Signaling Technology, 8553). Lysates were subjected to centrifugation at 12,000 g for 30 min at 4°C and protein concentrations were determined using the BCA protein assay reagent (Thermo Scientific). 50 μg of protein lysates were boiled for 10 min and subjected to SDS-PAGE electrophoresis using 4–15% precast gels (Bio-Rad, 567-1084). Densitometry was calculated using the Image Lab Software 5.2.1 (Bio-Rad). Antibodies used in this study are as follows: anti-frataxin (1:250, ab113691, Abcam), β-actin (1:1000, A2066, Sigma), goat anti-Rabbit HRP (1:5000, P0448, Dako) and goat anti-Mouse HRP (1:5000, P0447, Dako).
3
TRAIL and YM155 Combination Treatment Evaluation
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HeLa cells were treated with 25 ng/ml TRAIL, 25 nM YM155 or a combination of both for 24 h and subjected to western blot analysis. Cells were collected after 24-h incubation at 37°C and 5% CO2 and lysed with a protein extraction buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 2.5 mM sodium pyrophosphate, 1 mM b-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin) with a PMSF protease inhibitor (Cell Signaling Technology, Inc.). The protein concentration was determined by Bradford assay (Bio-Rad Laboratories, Inc.). Then, 50 µg protein was loaded on 8 or 12% SDS-PAGE and transferred to PVDF membrane (EMD Millipore) activated by methanol. Following transfer, non-specific binding was blocked with 5% skim milk and incubated at room temperature for 1 h. The membranes were probed with primary antibodies and incubated at 4°C for overnight. Subsequently, the membranes were incubated with a HRP-conjugated secondary antibody for 1 h at room temperature and washed thrice for 5 min with TBS-Tween 20 (0.05%) to remove unbound probes. The membranes were subjected to chemiluminescence-based detection (Pierce ECL Plus; Thermo Fisher Scientific Inc.). Imaging was performed using a ChemiDoc system with Image Lab software version 5.2 (Bio-Rad Laboratories, Inc.).
4
Extraction and Analysis of Cellular Fractions
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Cultured cells were washed once with cold PBS on ice. RIPA + 1 mM PMSF protease inhibitor (Cell-Signaling) was added and cells were carefully scraped within lysis buffer to extract extracellular matrix as well. Lysates were incubated at least 1 h on ice before remaining cell debris was removed by centrifugation and clear supernatant was stored at −80 °C. Nuclear and cytoplasmatic fractions were separated using the NE-PER extraction kit (Thermo Fischer) according to the manufacturer’s instructions. Immunoblot analysis was performed according to the manufacturer’s instructions, using NuPAGE 4-12% Bis-Tris Mini gels (Thermo Scientific) and semidry transfer on Nitrocellulose (GE Healthcare) membrane. Signal was detected via immunofluorescence using a LiCor Odyssey CXl or chemo-luminescence using ECL solution (Advansta) and photo films. All used primary and secondary antibodies are listed in Supplementary Table 2 .
5
Western Blot Analysis Protocol
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Cells were washed with ice-cold PBS and lysed in 1X Cell lysis buffer (Cell Signaling Technology, Cat. #9803S) containing 1mM PMSF protease inhibitor (Cell Signaling Technology, Cat. #8553S). Protein samples were prepared with NuPAGE LDS Sample Buffer (Thermo Fischer Scientific, Cat. #NP0007), NuPage Sample Reducing Agent (Thermo Fischer Scientific, Cat. #NP0004), and heated at 70 °C for 10 minutes. Samples were resolved by SDS-PAGE, transferred to nitrocellulose (Bio-Rad) and blocked in 5% milk/TBST for 1 h at room temperature. Membranes were incubated overnight with the respective primary antibodies at 4 °C, washed with 1X TBST, incubated with HRP-conjugated secondary antibodies and developed using SuperSignal™ West Dura Extended Duration Substrate (Thermo Fischer Scientific, Cat. #34076).
6
Comprehensive Cell Lysis and Protein Analysis
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Cells were washed with ice-cold PBS and lysed on ice for 30 min with cell lysis buffer containing RIPA buffer (Thermo Scientific, 89900) supplemented with 4 μg each of leupeptin (Sigma-Aldrich, L2884) and pepstatin (Sigma-Aldrich, P5318), 2 mM Na3VO4 (Sigma-Aldrich, 450243), 1 mM DL-Dithiothreitol (Sigma-Aldrich, 646653), 10 μM Calyculin A (Cell Signaling Technology, 9902), 250 μM β-glycerophosphate (Sigma-Aldrich, G9422) and 400 μM PMSF protease inhibitor (Cell Signaling Technology, 8553). Lysates were subjected to centrifugation at 12,000 g for 30 min at 4°C, and protein concentrations were determined using the Bradford assay (Bio-Rad, 5000006). Nuclear isolation for mature SREBP probing was performed using a Nuclear Extract Kit (Active Motif, 40010) with 10 μg/ml of the protease inhibitor ALLN (Millipore, 208719). Protein lysates were boiled for 10 min and subjected to SDS-PAGE electrophoresis using 4%–15% precast gels (Bio-Rad, 567-1084). Densitometry was calculated using the Image Lab Software 5.2.1 (Bio-Rad). Affinity purified custom antibodies for phosphorylated cPLA2 on Thr376 were developed by Thermo Fisher Scientific using the PLA2G4A-369:383 peptide antigen and used at a dilution of 1:500 in 5% BSA/TBST. All other primary antibodies were used at a dilution of 1:1000 in 5% BSA/TBST solution, and secondary antibodies at 1:5000 in 5% milk/TBST.
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