Total cell extracts were prepared using lysis buffer containing a cocktail of protease inhibitors (Carl Roth, Karlshuhe, Germany), as described previously [21 ]. Protein expression was analysed by Western blot [21 ]. The primary antibodies used were PAI1 (Becton Dickenson, Franklin Lakes, NJ, USA), uPA (Cusabio Technology LCC, Houston, TX, USA), and β-actin (Santa Cruz, Dallas, Texas, USA). Densitometric analysis was performed using the ImageJ program (version 1.52a, National Institute of Health, Bethesda, MD, USA).
Protease inhibitor
Manufactured by Carl Roth
Sourced in Germany
Protease inhibitor is a laboratory reagent used to inhibit the activity of proteases, which are enzymes that break down proteins. It helps preserve the integrity of protein samples during extraction, purification, and analysis procedures.
Automatically generated - may contain errors
Lab products found in correlation
Nuclease free water, by Promega (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Bradford assay method, by Carl Roth (2 mentions)
Chaps, by Carl Roth (2 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit, by Abcam (1 mentions)
Pvdf membrane, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Notch1, by Cell Signaling Technology (1 mentions)
Jagged1, by Santa Cruz Biotechnology (1 mentions)
Ikzf1, by Cell Signaling Technology (1 mentions)
Parp 1, by Santa Cruz Biotechnology (1 mentions)
Zymolyase 20t, by AMS Biotechnology (1 mentions)
Zymolyase, by AMS Biotechnology (1 mentions)
7 protocols using protease inhibitor
1
Protein Expression Analysis Protocol
2
Western Blot Analysis of EP1-4 Receptors in PMN- and M-MDSCs
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Proteins from isolated PMN- and M-MDSCs were extracted. In short, cells were lysed with NP-40 lysis buffer (150 mM NaCl, 1.0% NP-40, 50 mM Trist-HCl, protease inhibitors (Carl Roth)) for 30 minutes during continuous agitation and spun down at 16000 g for 20 minutes, after which the supernatant was collected. The cell lysates were separated by 10% SDS-PAGE gels in Laemmli loading buffer, and transferred to polyvinylidene fluoride (PVDF) membranes (Invitrogen, Carlsbad, CA, USA). After blocking with 5% skim milk/PBS/0.1% Tween (Carl Roth), blots were probed with primary antibodies for the EP1-4 receptors (Alomone Labs, Jerusalem, Israel, 1:250) and GAPDH (Abcam, Cambridge, UK, 1:2500). Membranes were then incubated with the appropriate secondary horseradish peroxidase-conjugated goat anti-rabbit (Abcam, 1:1000) for 1 hour at room temperature.
3
Comprehensive Protein Analysis via Western Blot
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Total cell extracts were prepared using lysis buffer containing a cocktail of protease inhibitors (Carl Roth, Karlsruhe, Germany), as described previously [12 ]. Proteins were analyzed by Western blot using chemiluminescence detection method [12 ]. Primary antibodies were used for detection of β actin, JAGGED1, PARP1, IKZF3 (all from Santa Cruz, Dallas, TX, USA), HES1, IKZF1, NOTCH1 and NOTCH1 cleaved (all from Cell Signaling Technology, Danvers, MA, USA). Densitometric analysis was performed using ImageJ program (NIH, Bethesda, MD, USA).
4
Extraction and Preservation of Hippocampal RNA and Proteins
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Fresh frozen hippocampal tissue specimens from mice and humans are stored at −80 °C until utilized for RNA extraction using peqGOLD TriFast™ (peqGOLD, Germany) according to the manufacturer's instructions to obtain RNA and proteins. The RNA pellet was extracted from the aqueous phase, air-dried, resuspended in 25 μl of nuclease-free water (Promega). The concentration of the RNA samples was measured using the Qubit 3.0 Fluorometer (High sensitivity, Invitrogen) and stored at −80 °C until further use. Proteins were extracted from the organic phase, cleaned with Ethanol, and the dried pellet resuspended with a 150 μl buffer containing 8M Urea in 4% (w/v) CHAPS and protease inhibitor (1:100 Carl Roth, Germany). The protein concentrations were determined using the Bradford assay method (Roth, Germany), and later the samples were stored until use at −80 °C.
5
Protein Aggregation Assay Protocol
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The assay used in this study was adapted from Koplin et al. (2010) (link). In brief, cell pellets were resuspended in zymolyase buffer (1.2 M sorbitol, 50 mM KPi, pH 7.2, 200 mM KCl2, 10 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride [PMSF]) containing 3 mg/ml zymolyase 20T (Amsbio, UK) and incubated for 30 min at 30°C. Washed cell pellets were resuspended in lysis buffer (0.5% Triton X-100, 50 mM KPi, pH 7.2, 10 mM DTT, 1 mM EDTA, 1× Protease inhibitor [Roth, Germany], 1 mM PMSF). After shaking for 20 min at 4°C, cleared supernatants were centrifuged at high speed (20,000 × g) for 20 min at 4°C. The pellet was washed once in wash buffer (0.5% Triton X-100, 20 mM KPi, pH 7.2, 1 mM PMSF) and subsequently washed in wash buffer without detergent. Aggregated proteins were analyzed by SDS–PAGE and Western blot. Aggregate pellet fractions were applied in threefold excess compared with total or supernatant fractions. Aggregation analysis in isolated mitochondria was performed as described above. For incubation of mitochondria at elevated temperatures, mitochondria were resuspended in buffer A (1.2 M sorbitol, 50 mM KPi, pH 7.2, 100 mM KCl, 10 mM MgCl2, 2 mM ATP, 2 mM NADH, 1 mM PMSF) before lysis.
6
Extraction of RNA and Proteins from Frozen Hippocampal Tissue
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Fresh frozen hippocampal tissue specimens from mice and humans are stored at -80°C until utilized for RNA extraction using peqGOLD TriFast TM (peqGOLD, Germany) according to the manufacturer's instructions to obtain RNA and proteins. The RNA pellet was extracted from the aqueous phase, air-dried, resuspended in 25μl of nuclease-free water (Promega). The concentration of the RNA samples was measured using the Qubit 3.0 Fluorometer (High sensitivity, Invitrogen) and stored at -80°C until further use. Proteins were extracted from the organic phase, cleaned with Ethanol, and the dried pellet resuspended with a 150 μl buffer containing 8M Urea in 4% (w/v) CHAPS and protease inhibitor (1:100 Carl Roth, Germany). The protein concentrations were determined using the Bradford assay method (Roth, Germany) and later the samples were stored until use at -80°C.
7
Extraction of RNA and Proteins from Frozen Hippocampal Tissue
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Fresh frozen hippocampal tissue specimens from mice and humans are stored at -80°C until utilized for RNA extraction using peqGOLD TriFast TM (peqGOLD, Germany) according to the manufacturer's instructions to obtain RNA and proteins. The RNA pellet was extracted from the aqueous phase, air-dried, resuspended in 25μl of nuclease-free water (Promega). The concentration of the RNA samples was measured using the Qubit 3.0 Fluorometer (High sensitivity, Invitrogen) and stored at -80°C until further use. Proteins were extracted from the organic phase, cleaned with Ethanol, and the dried pellet resuspended with a 150 μl buffer containing 8M Urea in 4% (w/v) CHAPS and protease inhibitor (1:100 Carl Roth, Germany). The protein concentrations were determined using the Bradford assay method (Roth, Germany) and later the samples were stored until use at -80°C.
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