LN229 or HEK293T cells expressing indicated recombinant proteins were rinsed in ice‐cold PBS and lysed in RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 4 mM EDTA, 1 mM EGTA, 1% Triton X‐100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol, 1x phosphatase inhibitor (Roche), and 1x proteinase inhibitor (Roche)) or RIPA buffer without SDS as indicated. Cell extracts were incubated with benzonase nuclease (Sigma #E1014) at 4 °C for 30 min. After spinning down, the supernatants were incubated with anti‐FLAG M2 affinity gel (Sigma) at 4 °C for 2 h. The beads were washed for three times with IP2 buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 4 mM EDTA, 1 mM EGTA, 0.5% NP‐40, 10% glycerol, 1x phosphatase inhibitor (Roche) and 1x proteinase inhibitor (Roche)) and eluted with 3xFLAG Peptide (Sigma # F4799) dissolved in TBS (10 mM Tris HCl, 150 mM NaCl, pH 7.4). The proteins were then prepared and separated on 4–12% Bis‐Tris SDS‐PAGE gels (Invitrogen) and subjected to standard immunoblotting.
Proteinase inhibitor
Manufactured by Roche
Sourced in United States, Switzerland, Germany, China, Spain, Macao
Proteinase inhibitors are a class of laboratory reagents used to prevent the degradation of proteins in biological samples. They work by inhibiting the activity of proteolytic enzymes, which can break down proteins. Proteinase inhibitors are commonly used in protein extraction, purification, and analysis workflows to preserve the integrity of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Roche (21 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (16 mentions)
Ripa buffer, by Merck Group (13 mentions)
Bca kit, by Thermo Fisher Scientific (12 mentions)
Pvdf membrane, by Merck Group (11 mentions)
Nitrocellulose membrane, by Bio-Rad (11 mentions)
Ripa buffer, by Thermo Fisher Scientific (9 mentions)
Rnalater, by Thermo Fisher Scientific (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (7 mentions)
β actin, by Merck Group (5 mentions)
Ripa lysis buffer, by Beyotime (5 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (5 mentions)
Ripa lysis buffer, by Merck Group (5 mentions)
β actin, by Cell Signaling Technology (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (5 mentions)
Dissecting microscope, by Canon (4 mentions)
10 μl pipettor, by Eppendorf (4 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (4 mentions)
Phosphatase inhibitor, by Merck Group (4 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (4 mentions)
217 protocols using proteinase inhibitor
1
Affinity Purification of FLAG-Tagged Proteins
2
Isolation of Nuclear Fraction from HEK Expi293 Cells
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HEK Expi293 cells were harvested by centrifugation and washed two times with ice-cold phosphate-buffered saline (PBS). One milliliter of pellet was suspended with 2 ml of ice-cold buffer A [10 mM Hepes (pH 7.4), 5 mM MgCl2, 250 mM sucrose, 1× proteinase inhibitor (Roche), and 0.5 mM DTT] and spun down at 500g for 5 min at 4°C. Afterward, the pellet was resuspended in 5 ml of ice-cold buffer A for 15 min on ice, supplemented with NP-40 (final concentration, 0.1%) on ice for 5 min, and spun down at 1000g for 5 min at 4°C. Then, the pellet was resuspended with 2 ml of ice-cold buffer A and spun down at 1000g for 5 min at 4°C. The pellet was saved as the nuclear fraction. The nuclei were resuspended with 0.75 ml of buffer B [20 mM Hepes (pH 7.4), 1.5 mM MgCl2, 20% glycerol, 0.5 mM EDTA, 1× proteinase inhibitor (Roche), 0.5 mM DTT, and 0.42 M NaCl] to incubate for 45 min at 4°C. The mixture was ultracentrifuged in a Beckman SW 40 Ti rotor at 40,000 rpm for 90 min at 4°C. The supernatant was saved as nuclear extract for further density gradient centrifugation.
3
Isolation and Characterization of Cellular Organelles
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Adapted from Malchow et al. [65 (link)], vegetatively growing cells were washed twice with KK2 buffer (19 mM KH2PO4, 3.6 mM K2HPO4); 3.5 × 108 cells were resuspended at a density of 1.4 × 107 cells/ml in KK2 plus 5 mM EGTA and shaken at 120 rpm for 4 h, pulsing with exogenous cAMP (50 nM every 6 min). Cells were washed with ice-cold 20 mM HEPES, pH 7.2, twice and resuspended in 4 ml of this buffer containing 1× proteinase inhibitor; Roche. Samples were disaggregated using 21-G needles then homogenized by passage through 3-μm nucleopore filters (Millipore). Homogenate was added to 4 ml of 2× buffer 1 (100 mM KCl, 2 mM MgCl2, 6% sucrose, 2× proteinase inhibitor, Roche). After centrifugation for 5 min at 200g to remove residual intact cells, P0 was obtained by centrifugation at 3800g for 5 min and resuspended in 30 μl buffer 2 (10 mM Tris HCl, 50 mM KCl, 1 mM MgCl2, 3% sucrose, 2× proteinase inhibitor). P1 was obtained by centrifugation at 12,000g for 20 min and resuspended in 25 μl of buffer 2. P1 vesicles contained a mixture of markers of both the endoplasmic reticulum (calnexin) and acidic contractile vacuole (dajumin). All the experiments described here were performed on P1 vesicles. All procedures were carried out at 4 °C. Samples were either used fresh or aliquots snap frozen on dry ice and stored at − 80 °C up to 7 days.
4
Mesenteric Artery Preparation for EM
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Mesenteric resistance arteries were dissected, open longitudinally and denuded from endothelium by incubation in 50 mM Tris–HCl plus 20 mM EDTA, pH 7.4, plus proteinase inhibitors (Roche), followed by a second incubation in 50 mM Tris–HCl, 1 M NaCl, pH 7.4, plus proteinase inhibitors. Arteries were then processed for electron microscopy as described (Maser & Trimble, 1977 ). Samples were visualized and imaged with a Hitachi FESEM S5000, 20 kV.
5
Western Blot Analysis of Protein Expression
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Cells (1 × 106) were collected and washed with PBS three times. The total protein was extracted by lysis buffer (Beyotime) containing 1X proteinase inhibitor (Roche) for 30 minutes on ice. The supernatants were collected by centrifugation. 20 μg of protein was loaded on SDS-PAGE (12.5%) and transferred onto the PVDF membranes. After being blocked by notfat milk, diluted primary antibodies and relevant HRP conjugated secondary antibodies were incubated, ECL detection reagents and X-ray film espousing were used to detect the protein expression. The GOLM1, B7-H3 antibody (Abcam), actin, VEGF, MMP-9 antibody (Santa Cruz), and E-cadherin antibody (CST) were used according to the manufacturer's recommendations.
6
Production and Purification of GntR-His
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To produce C-terminally hexahistidine-tagged Sco1678 protein (GntR-His), its ORF was cloned into pET28a expression vector using PCR and primers SCO1678NcoI-f and SCO1678HindIII-r. Resulting plasmid was labeled as pET28a-SCO1678. For GntR-His production, E. coli BL21 (DE3) GOLD carrying pET28a-SCO1678 was grown in LB supplemented with tetracycline and kanamycin until OD600 reached 0.5; then the culture was induced with 1 mM IPTG (isopropylthiogalactoside) and incubated for six hours at 22°C. Cells were collected by centrifugation and resuspended in a lysis buffer (50 mM Na2HPO4, 300 mM NaCl, and 20 mM imidazole, pH 7) containing proteinase inhibitor (Roche). Cells lysis was achieved by two consecutive passages through a French press (American Instrument Corporation) at 1000 psi. The cell lysate was centrifuged at 18000 rpm for 30 minutes and soluble fraction was applied to Ni-NTA agarose resin (Qiagen), washed two times with wash buffer (50 mM Na2HPO4, 300 mM NaCl, and 40 mM imidazole, pH 7). The protein was eluted with 200 mM imidazole and dialyzed against storage buffer (50 mM Na2HPO4, 300 mM NaCl, and 5% glycerol, pH 7). Protein concentration was determined by Bradford assay.
7
Western Blot Analysis of EMT Markers
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RIPA lysis buffer (Sigma, USA) supplemented with proteinase inhibitor (Roche Applied Science, Indianapolis, IN, USA) was managed to extract total protein, whose concentration was determined via BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Exactly 30 μg protein extracts were electrophoresed within 6%‐10% polyacrylamide gel and were then transferred onto the PVDF membrane. Subsequently, the membranes were blocked at room temperature for 1 hour, and we added rabbit anti‐human monoclonal antibodies for E‐cadherin, vimentin, and N‐cadherin (1:1000, Proteintech, Manchester, UK), along with rabbit anti‐human GAPDH monoclonal antibody (1:1000, CST, Danvers, MA, USA). After that, corresponding mouse anti‐rabbit secondary antibodies for E‐cadherin, vimentin, and N‐cadherin (1:10 000, CST) were supplemented for 1‐hour culture at room temperature. Finally, ECL detection reagent (Millipore, Billerica, MA, USA) was applied for development, and images were analyzed with Gene Genius gel imaging system (Syngene, Cambridge, UK).
8
Western Blotting of Signaling Proteins
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Cells were lysed on ice with radioimmunoprecipitation assay (RIPA) buffer containing a proteinase inhibitor (Roche) and a phosphatase inhibitor cocktail (Sigma‐Aldrich). Immunoblotting was performed using antibodies against MIG‐6 (Cell Signaling Technology, Cat. #2440), GLUT1 (Abcam, clone SPM498), p‐EGFR (Tyr1173) (Cell Signaling Technology, clone 53A5), EGFR (Santa Cruz Biotechnology, clone 1005), pAKT(S473) (Cell Signaling Technology, Cat.9271), AKT (Cell Signaling Technology, clone C67E7), HIF1α (BD Biosciences, Clone 54), cMyc (Roche, clone 9E10), β‐actin (Sigma‐Aldrich, clone AC‐15), and α‐Tubulin (Sigma‐Aldrich, clone B‐5‐1‐2). Detailed antibody information is listed in Table EV4 .
9
Western Blot Protein Analysis Protocol
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Cell proteins were prepared using a cell lysate solution (KeyGen Biotech, Nanjing), containing 10% proteinase inhibitor (Roche, Basel, Switzerland). Proteins were separated on 10% to 12% SDS-polyacrylamide gels and electrophoretically transferred onto polyvinylidene fluoride membranes. The membranes were incubated overnight at 4 °C with primary antibodies. The next day, the membranes were incubated with secondary antibodies at room temperature for 1 hour. Signals were detected by ECL Plus (Millipore) and captured by X-ray films (Fuji, Shizuoka). The films were scanned with a GS-800 Calibrated Densitometer (Bio-Rad), and the densitometry was analyzed using the Quantity One software.
10
Tissue Homogenization for Protein Analysis
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Each sample was mixed with 200 µL SEID buffer (300 mM sucrose, 20 mM EDTA, 100 mM imidazole, 0.1% sodium deoxycholate, pH 7.5) containing proteinase inhibitor (#11836145001; Roche, Indianapolis, IN, USA; v/v: 25/1) and then homogenized on ice. The homogenate was centrifuged at 5,000 × g at 4°C for 5 minutes. The supernatants were collected and used for immunoblotting, NKA activity assays, or protein concentration analysis. Protein concentrations were determined using a BCA protein assay kit (#23225; Pierce) with bovine serum albumin (#23209; Pierce) as a standard.
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