To investigate the expression of target proteins, total proteins were determined by Western blotting assay. Proteins were extracted using Pierce RIPA buffer (Applygen, Beijing, China) supplemented with protease phosphatase inhibitor (Beyotime, Shanghai, China). To collect total proteins, the cell lysates were removed by centrifugation at 12,000 RPM at 4°C for 20 min. Total proteins were quantified using the bicinchoninic acid (BCA) protein assay kit (Beyotime, Shanghai, China) according to the manufacturer’s instructions. The total proteins were further separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck-Millipore, Darmstadt, Germany). The blots on PVDF membranes were blocked with 5% BSA in tris-buffered saline with Tween 20 (TBS-T) solution, incubated with the appropriate primary antibodies at 4°C overnight, and further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature (25°C) for 1 h. Afterward, the blots were visualized with Immobilon Western chemiluminescent HRP substrate (Millipore, Burlington, MA, USA), and images were obtained using MiniChemi610 (Sage Creation, Beijing, China). The quantification of blots was performed by ImageJ.
Protease phosphatase inhibitor
Manufactured by Beyotime
Sourced in China
Protease/phosphatase inhibitors are chemical compounds used in laboratory settings to prevent the degradation of proteins by proteases or the dephosphorylation of proteins by phosphatases. These inhibitors help maintain the integrity and stability of protein samples during various experimental procedures.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (7 mentions)
Pvdf membrane, by Merck Group (6 mentions)
Radioimmunoprecipitation assay buffer, by Beyotime (3 mentions)
Immobilon psq membrane, by Merck Group (2 mentions)
Loading buffer, by Beyotime (2 mentions)
Bca assay kit, by Beyotime (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Minichemi610, by Sagecreation (1 mentions)
Immobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
Chemidoc xrs gel imaging system, by Bio-Rad (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Ab32515, by Abcam (1 mentions)
Ab32096, by Abcam (1 mentions)
Ab112490, by Abcam (1 mentions)
Ab203490, by Abcam (1 mentions)
Ab136918, by Abcam (1 mentions)
Ab64693, by Abcam (1 mentions)
Ab6721, by Abcam (1 mentions)
20 protocols using protease phosphatase inhibitor
1
Western Blot Protein Expression Analysis
2
Western Blot Analysis of AKT/PI3K Pathway
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Cells or ABs were lysed in RIPA lysis buffer (CWBIO, Beijing, China) mixing with protease phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). The lysis system was centrifuged at 12,000 × g for 15 min to obtain the supernatant after incubated on ice for 30 min. The BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China) was used to detect the concentration of proteins. 50 μg protein samples were diluted in loading buffer (Beyotime Biotechnology, Jiangsu, China) and electrophoresed, followed by transferred onto polyvinylidene fluoride membranes (ImmobilonTM-PSQ Membranes, Sigma-Aldrich, China) and blocked in 5% bovine serum albumin (BSA). Protein on membranes were incubated with anti AKT, anti p-AKT, anti PI3K, anti p-PI3K, anti S6K, anti p-S6K and anti β-actin (1:1000, diluted in 5% BSA) antibodies for 12 h at 4 °C. After washing in Tris Buffered Saline Tween buffer three times, secondary antibodies linked HRP (1:10,000, diluted in 5% BSA) against primary antibodies were used to incubate membranes. Super ECL Plus (Biosharp, Anhui, China) and the ChemiDoc XRS + gel imaging system (BioRad) were used to detect chemiluminescent signals.
3
Western Blot Analysis of Neuroinflammatory Markers
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Hippocampus tissues were lysed in radio-immunoprecipitation assay buffer (Beyotime) containing 1% protease/phosphatase inhibitor (Beyotime). The protein samples were harvested after centrifugation at 10, 000 × g for 5 min, and quantified with a bicinchoninic acid (BCA) assay kit (Beyotime). The samples (20 μg) were separated via sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred onto polyvinylidene fluoride membranes (Bio-Rad, Hercules, CA, USA). After blocking in 3% bovine serum albumin (Solarbio, Beijing, China), the membrane was incubated with anti-BDNF (ab108319, 1:1000 dilution, Abcam), anti-NGF (ab52918, 1:500 dilution, Abcam), anti-iNOS (ab136918, 1:2000 dilution, Abcam), anti-CD86 (ab112490, 1:500 dilution, Abcam), anti-Arg-1 (ab203490, 1:1000 dilution, Abcam), anti-CD206 (ab64693, 1:500 dilution, Abcam), anti-ERK (ab184699, 1:1000 dilution, Abcam), anti-p-ERK (ab201015, 1:500 dilution, Abcam), anti-CREB (ab32515, 1:500 dilution, Abcam), anti-p-CREB (ab32096, 1:1000 dilution, Abcam), or anti-β-actin (ab20272, 1:5000 dilution, Abcam) antibody overnight at 4°C. Membranes were then incubated with HRP-conjugated IgG (ab6721, 1:5000 dilution, Abcam) for 2 h. β-actin acted as a control. After exposure to the ECL Western Blotting Substrate (Solarbio), the blots were analyzed utilizing Quantity One software (Bio-Rad).
4
Quantifying Protein Expression Changes
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Changes in the expression of the following proteins, such as ITGA5, FAK, and AKT, were detected by Western blotting. Proteins were lysed using RIPA lysis buffer (Thermo Fisher Scientific) and protease/phosphatase inhibitor (Beyotime Biotechnology), and their concentrations were detected by BCA (Thermo Fisher Scientific). Proteins were separated by 10% polyacrylamide gel electrophoresis, transferred to a PVDF membrane (GE Healthcare, Piscataway, NJ), and blocked with 5% skimmed milk powder for 1 h at room temperature. The membrane was then incubated overnight with primary antibodies on a 4°C shaker. The antibodies and concentrations used were the following: ITGA5 (Proteintech, 1 : 1000 dilution), FAK (Cell Signaling Technology, 1 : 1000 dilution), p-FAK (Cell Signaling Technology, 1 : 2000 dilution), AKT (Cell Signaling Technology, 1 : 1000 dilution), p-AKT (Cell Signaling Technology, 1 : 1000 dilution), and GAPDH (Cell Signaling Technology, 1 : 10000 dilution). Next, the membrane was incubated with the secondary antibody (Cell Signaling Technology 1 : 5000 dilution) at room temperature for 1 hr. Finally, the membrane was treated with ECL developer (Pierce), and the bands were developed using a gel imager FluorChem (USA, ProteinSimple).
5
Protein Extraction and Western Blot Analysis
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The protein samples were centrifuged at 4°C, and tissues were homogenized in radioimmunoprecipitation assay buffer (Beyotime) containing phenylmethylsulfonyl fluoride and protease/phosphatase inhibitor (Beyotime). Thereafter, 5 × loading buffer was added to the protein supernatant, and total protein was quantified using BCA analysis kits (Beyotime). Non-specific protein binding on membranes was blocked with 5% skimmed milk for 2 h, then the membranes were incubated with primary antibody overnight at 4°C, followed by secondary antibody at 37°C for 60 min. Chemiluminescence was detected using Enhanced Chemiluminescence Kits and a gel imaging system (Invitrogen). The density of protein bands was determined using ImageJ software.
6
Evaluating PD1 Gene Recombinant Exosomes
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For identifying PD1 gene recombination engineered exosomes, Western blot assay was performed to evaluate the PD protein expression. Briefly, Exo, PD1 Exo, PD1-Imi Exo or gene recombinant HEK 293T cells were collected, and lysed in RIPA buffer (Applygen, Beijing, China) with protease phosphatase inhibitor (Beyotime, Shanghai, China) at 4 °C for 1 h. The lysed mixture was then centrifuged at 12000 rpm at 4 °C for 20 min for collecting its supernatant. Afterwards, the protein concentration in the supernatant was quantified by BCA assay kit, and further separated by 4%–20 % precast sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The separated proteins were transferred onto polyvinylidene fluoride (PVDF) membranes (Merck-Millipore, Darmstadt, Germany) and blocked with 5 % bovine serum albumin (BSA) in TBS-T (Tris buffered saline with Tween 20) solution. After incubating with the appropriate primary antibodies at 4 °C overnight, the PVDF membranes was further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies at 25 °C for 1 h. Finally, the blots were observed by a MiniChemi610 imaging system (Sage Creation, Beijing, China).
7
Western Blot Analysis of LUAD Tissues
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Western blot analysis was performed as previously described [31 (link)]. LUAD tissues and cell lines were lysed in RIPA buffer containing protease/phosphatase inhibitor (Beyotime, China). The samples were grinded with a mechanical homogenizer and centrifuged for 15 min, 12,000 rpm at 4 °C. Protein samples were further electrophoresed on corresponding concentration of SDS-PAGE gels and transferred to PVDF membranes (Millipore, Eschborn, Germany). The membrane was blocked for 2 h at room temperature with 5% milk in Tris-buffered saline (TBS) with 0.1% Tween 20 (Biofroxx, Germany) before incubating with primary antibody overnight at 4 °C. Subsequently, an appropriate HRP-conjugated secondary antibody was incubated for 1 h at room temperature. Western blot was visualized with chemiluminescence reagents (Biosharp, China).
8
Western Blot Analysis of Brain Protein
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Western blot was performed as previously described (28 (link)). Brains were weighed and homogenized in ice cold RIPA buffer (Beyotime, Shanghai, China) containing 1% protease phosphatase inhibitor (Beyotime, Shanghai, China), followed by centrifugation at 10,000 g for 15 min at 4°C to collect the supernatant. The protein concentration was determined by the BCA protein assay reagent (Tiangen Biotech, Beijing, China). The samples were stored at −20°C. After separation by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were transferred to activated polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, United States). Membranes were blocked in 5% skim milk for 1 h at room temperature, and then incubated with primary antibodies targeting Iba1 (Abcam, ab178846, 1:1,000) and β-actin (Bioss, #bs-0061R, 1:1,000) overnight at 4°C. After being washed with TBST solution for 5 times, membranes were incubated with the horseradish-peroxidase-conjugated secondary antibody for 1 h at room temperature. The bands were visualized by enhanced chemiluminescence reagent (Millipore, Bedford, MA, United States) and quantified using ImageJ software.
9
Protein Extraction and Immunoblotting Protocol
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Cells or sEVs were lysed in RIPA lysis buffer mixing with protease phosphatase inhibitor (Beyotime Biotechnology, Jiangsu, China). The lysis system was incubated on ice for 30 mins, and ultrasonic lysed every 10 mins. Subsequently, the supernatant was carefully removed and transferred into a new microfuge tube after centrifugation at 13000 g for 15mins. The BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China) was used to detect the concentration of proteins according manufacturer's instructions. Each sample (50 μg) was diluted in loading buffer (Beyotime Biotechnology, Jiangsu, China) and subjected to a standard SDS-PAGE followed by transferred onto polyvinylidene uoride membranes (ImmobilonTM-PSQ Membranes, Sigma-Aldrich, China). After blocking in 5% skim milk and washing in Tris Buffered Saline Tween buffer, proteins were detected using the following antibodies: Histone 3 (bs-17422R) at a 1:1000 dilution, CD81 (bs-2489R) at a 1:1000 dilution, TSG101 (bs-1365R) at a 1:1000 dilution, LaminA/C (bs-1839R) at a 1:1000 dilution, and β-actin (bs-0061R) at a 1:2000 dilution. Corresponding secondary antibodies against primary antibodies were used by an hour of incubation (1:2000) . Blots against β-actin served as loading control. Super ECL Plus (Bioground, Chongqin, China) and Immun-Star HRP (BioRad) were used to detect chemiluminescent signals.
10
Protein Extraction and Western Blot Analysis
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After treatment, cells were collected, and the total proteins were extracted using RIPA solution (Servicebio, Wuhan, China) containing a mixture of protease phosphatase inhibitors (Beyotime, Nanjing, China). A BCA protein assay kit was used to measure the total concentrations of proteins according to the manufacturer’s instructions. Twenty micrograms of cellular protein from each group was electro-blotted onto a PVDF membrane (Millipore, Burlington, MA, USA) following separation on 10% SDS-PAGE (Sangon Biotech, Shanghai, China). The membranes were blocked with 5% BSA in 0.1% Tween 20/Tris-buffered saline (TBST) for 2 h at room temperature and then incubated with either anti-BACH1 ((F-9): sc-271211) or anti-HOMX1 (10701-1-AP) antibodies overnight at 4 °C. Afterwards, samples were washed thrice with 0.1% TBST and incubated with secondary antibodies (1:2000) (Beyotime, Nanjing, China) at room temperature for 2 h. The enhanced chemiluminescence kit (Cat No: WBKLS0500) was used to visualize the protein bands.
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