Cells were lysed in PRO-PREPTM protein extraction solution (iNtRON Biotechnology, Cat# 17081) supplemented with phosphatase inhibitor cocktail (Gen DEPOT, Cat. No. p3200-001). Protein concentrations were then measured using a Bio-rad Bradford assay (Cat# 500-0006, Hercules, CA, USA). Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Cat# 10600023). The membranes were blocked with Tris-buffered saline containing 0.05% Tween 20 and 5% non-fat dry milk for 1 h, and membranes were incubated overnight at 4 °C with primary antibody. HER2 (Cat# 2165, RRID:AB_10692490), p-HER2 (Cat# 2247, RRID:AB_331725), p-EGFR (Cat# 2234, RRID:AB_331701), Akt (Cat# 9272, RRID:AB_329827), p-Akt (Cat# 9271, RRID:AB_329825), ERK (Cat# 9102, RRID:AB_330744), and p-ERK (Cat# 9101, RRID:AB_331646) antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA), EGFR (Cat# sc-03, RRID:AB_631420) was obtained from Santa Cruz, and ß-actin (Cat# A5316, RRID:AB_476743) was obtained from Sigma (Saint Louis, MO, USA). Then, horseradish peroxidase-conjugated secondary antibodies were incubated for 1 h at room temperature. Immunoreactive bands were detected with chemiluminescence reagent (Cat# RPN2106).
Phosphatase inhibitor cocktail
Manufactured by GenDEPOT
Sourced in United States, Germany
Phosphatase inhibitor cocktail is a solution that contains a mixture of chemical compounds designed to inhibit the activity of phosphatases, which are enzymes that remove phosphate groups from proteins. This product is commonly used in biochemical and cell biology research applications to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by GenDEPOT (8 mentions)
Bca protein assay, by Thermo Fisher Scientific (4 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P erk, by Cell Signaling Technology (2 mentions)
Bca protein kit, by Bio-Rad (2 mentions)
Ibind automated western system, by Thermo Fisher Scientific (2 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
M00660, by GenScript (2 mentions)
Las4000, by GE Healthcare (2 mentions)
Gapdh, by Santa Cruz Biotechnology (2 mentions)
Bradford assay kit, by Bio-Rad (2 mentions)
Dc protein assay reagents package, by Bio-Rad (2 mentions)
Lf qc0101, by AbFrontier (2 mentions)
Trizol, by Molecular Research Center (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
24 protocols using phosphatase inhibitor cocktail
1
Western Blot Protocol for Protein Expression
2
Protein Extraction and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with RIPA buffer (50 mM Tris-HCl, pH 7.4; 1% Nonidet P-40; 0.25% sodium deoxycholate; 150 mM NaCl; 1 mM Na3VO4) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride; 100 μg/mL leupeptin; 10 μg/mL pepstatin, 1 μg/mL aprotinin; 2 mM EDTA) and a phosphatase inhibitor cocktail (GenDEPOT, Baker, TX, USA). After incubation for 20 min at 4 °C, the lysates were centrifuged at 13,000 rpm for 20 min at 4 °C, and the supernatants were collected for western blotting. Protein concentration was measured using a BCA protein kit (Bio-Rad, Hercules, USA). The lysates were subjected to SDS-PAGE gel electrophoresis, transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA), and incubated with 5% skimmed milk for 1 h at room temperature. The membranes were then probed with primary antibodies against α-SMA (Abcam, Cambridge, MA, USA), FPR2 (Novus Biological, Littleton, CO, USA), and actin (Santa Cruz Biotechnology, Dallas, TX, USA), followed by incubation with horseradish peroxidase-coupled secondary antibodies (Jackson Immunoresearch, West Grove, PA, USA). Proteins were detected using ECL Plus western blotting detection reagents (Amersham Biosciences, Piscataway, NJ, USA).
3
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MIAPaCa-2 and PANC-1 cells were washed with Dulbecco’s phosphate buffered saline (DPBS) and lysed with RIPA buffer (Biosesang, Korea) containing 1% Triton X-100, Xpert protease inhibitor, and phosphatase inhibitor Cocktail (GenDEPOT, TX, USA). Proteins were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, MA, USA). Protein transfer was verified using the Ponceau S staining solution (Amresco, OH, USA), and the blots were then incubated with the appropriate primary (1:500, except for β-actin [1:10,000]) and the secondary antibodies (1:1,000, except for β-actin [1:20,000]) conjugated to HRP. Antibody binding was detected using an enhanced chemiluminescence reagent (Bio-Rad, CA, USA) using primary antibodies specific to the proteins of interest, and the proteins were detected using X-ray film and enhanced chemiluminescence reagent. Primary antibodies were used against the following: p-ERK, ERK, p-AKT, AKT, and β-actin (Cell Signaling Technologies, MA, USA) and p-BRAF (Santa Cruz Biotechnology, TX, USA), and the secondary antibodies were purchased from Cell Signaling Technologies.
4
Isolation and Detection of O-GlcNAcylated Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Agarose-bound sWGA was purchased from Vector Laboratories (#AL-1023S; USA). The sWGA pulldown assay was performed according to the manufacturer’s instructions. O-GlcNAcylated proteins were eluted with elution buffer containing 0.1 M N-acetyl glucosamine at room temperature for 20 min. Western blot analysis was performed according to a modified version of a previously described method24 (link). Cells were lysed with lysis buffer containing a protease inhibitor cocktail (P3100; genDEPOT, USA) and a phosphatase inhibitor cocktail (P3200; genDEPOT). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein transfer, the target protein was detected using the iBind™ Automated Western system (Thermo Fisher Scientific, USA). Anti-FLAG (#F1804) and anti-ActB (#AC-74) antibodies were purchased from Sigma-Aldrich. Anti-Sox2 (ab97959) antibody was purchased from Abcam (UK), and anti-Oct4 (sc-5279) antibody was purchased from Santa Cruz Biotechnology (USA).
5
Protein Expression Analysis in Cells and Mouse Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells and mouse brains were lysed on ice in RIPA buffer (50 mM Tris–HCl, pH 7.4, 1 % NP-40, 0.25 % Na-deoxycholate, 150 mM NaCl, 1 mM Na3VO4, and 1 mM NaF) containing protease inhibitors (2 mM phenylmethylsulfonyl fluoride [PMSF], 10 μg/ml leupeptin, 10 μg/ml pepstatin, and 2 mM EDTA) and a phosphatase inhibitor cocktail (GenDEPOT, Barker, TX, USA). Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and identified using specific antibodies. The antibodies for PINK1 (Cat. NO. 23707), MAP2, and MBP were obtained from Abcam (Cambridge, MA, USA); for Parkin, p-SMAD1/5/8, and p-STAT3 from Cell signaling technology (Danvers, MA, USA); for nestin and CNPase from Millipore (Bedford, MA, USA); for TUJ-1 from Covance (Berkeley, CA, USA); for GFAP from Sigma (Cat. No. G3893; St. Louis, MO, USA); and for HES1 and GAPDH from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Membranes were incubated with peroxidase-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA), and visualized with an enhanced chemiluminescence system (Daeil Lab Inc., Seoul, Korea).
6
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein were extracted from cells using RIPA buffer (GenDEPOT, TX, USA) containing 1% protease inhibitor and 1% phosphatase inhibitor cocktail (GenDEPOT) as lysis buffer. In total, 20 μg of extracted proteins is separated by SDS-PAGE and transferred to a nitrocellulose membrane (GE Healthcare, Chalfont St Giles, UK). Immunoblotting was performed as previously described by Gyamfi et al.4 (link) List of all primary antibodies and dilutions used are indicated in Supplementary Table 6 . All gels and blots were derived from the same experiment and were processed in parallel.
7
Immunoprecipitation and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The transfected cells were washed in chilled PBS three times and lysed directly using cell lysis buffer (#9803, Cell Signaling Technology), including 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, pH7.5 for 30 min at 4°C. The lysis buffer contained 1X protease inhibitor cocktail (P3100-010, GenDEPOT) and phosphatase inhibitor cocktail (P3200-001, GenDEPOT) to prevent degradation and removal of post-translational modifications. The lysates were clarified by centrifugation at 20,000 g at 4°C for 10 min. The supernatant was collected, with the total protein concentrations determined by a BCA protein assay (#23225; Thermo Scientific). For immunoprecipitation experiments, equivalent sample amounts were incubated with the magnetic Streptavidin C1 beads (#65001, Thermo Scientific) overnight at 4°C. The beads were then pelleted and washed with lysis buffer five times. 1X denaturing loading buffer was added and heated for 5 min at 95°C before loading into SDS-PAGE. Cell lysates were electrophoretically separated on 8–16% SDS-PAGE (M00660, GenScript), followed by transferring to PVDF membranes and probing with appropriate antibodies.
8
Immunoblot and Immunoprecipitation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For immunoblot analysis, cells were lysed with RIPA lysis buffer (20 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP-40, and 1 mM EDTA) containing protease inhibitor cocktail (GenDepot, P3100-001) and phosphatase inhibitor cocktail (GenDepot, P3200-001). Whole-cell lysates were subjected to SDS-PAGE followed by immunoblotting with the indicated antibodies. To detect phosphorylated EPRS1 proteins, cells were lysed with Phosphosafe extraction buffer (Millipore, #71296) containing protease inhibitor at 4 °C. Immunoprecipitation was performed with cell lysates that had been pre-cleared with protein A/G beads for 1 h at 4 °C. The pre-cleared cell lysates were incubated overnight with the indicated antibodies at 4 °C, followed by incubation with 30 μl protein A/G PLUS-agarose beads for 3 h at 4 °C. The immunoprecipitates were collected and washed extensively with lysis buffer before immunoblot analysis.
9
Cell Lysis and Immunoprecipitation Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells were washed in chilled PBS three times and lysed directly using a CST lysis buffer (20 x 10–3 m Tris‐HCl (pH 7.5), 150 x 10–3m NaCl, 1 x 10–3m EDTA, 1 x 10–3m EGTA, 1% Triton X‐100, 2.5 x 10–3m Na pyrophosphate, and 1 x 10–3m β‐glycerophosphate, pH7.5) for 30 min at 4 °C. The lysis buffer contained 1X protease inhibitor cocktail (P3100‐010, GenDEPOT) and phosphatase inhibitor cocktail (P3200‐001, GenDEPOT). After centrifugation at 20 000 g at 4 °C for 10 min, the supernatant was collected, with the total protein concentrations determined by a BCA protein assay (#23225, Thermo Scientific). For immunoprecipitation, the cell lysates were incubated with the indicated antibodies and magnetic A/G beads (#88803, Thermo Scientific) overnight at 4 °C. The beads were then pelleted and washed with lysis buffer five times. 1X denaturing loading buffer was added and heated for 10 min at 95 °C before loading into SDS‐PAGE gels. Cell lysates were electrophoretically separated on 8–16% SDS‐PAGE (M00660, GenScript), followed by transfer onto a nitrocellulose blotting membrane (A15735264, GE Healthcare, Life science) and probed with appropriate antibodies.
10
Protein Extraction and Western Blotting Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed with PBS and lysed in ice-cold RIPA buffer (50 mM Tris-HCl [pH7.4], 150 mM NaCl, 0.25% sodium deoxycholate and 1% Triton X-100) containing protease inhibitor cocktail (Calbiochem, Germany) and phosphatase inhibitor cocktail (GenDEPOT, Baker, TX). After brief sonication, the lysates were incubated on ice for 20 min and centrifugated at 15,700g for 30 min at 4 ℃. After centrifugation, the supernatant was collected. The protein concentrations were determined with a bicinchoninic acid (BCA) protein assay kit (Bio-Rad, CA, USA). Proteins were separated by SDS-PAGE and transferred to PVDF membranes. The PVDF membrane was blocked with 1% BSA in Tris-buffered saline. After 1 h blocking, the membrane was immunoblotted with appropriate antibodies and visualized using an enhanced chemiluminescence (ECL) system (Thermo Fisher Scientific, Waltham, MA).
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!