Total proteins were extracted from B-CPAP cells using radioimmunoprecipitation assay and quantified using a bicinchoninic acid assay kit (Solarbio). In brief, 20 μg of harvested proteins from each group was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. The membranes were then blocked with 5% skim milk at 4°C overnight and cultured for 1 h with primary antibodies against B cell lymphoma-2 (Bcl-2), Bcl-2/Bcl-XL-associated death promoter (Bad), lactate dehydrogenase A (LDHA), hexokinase 2 (HK2), Akt (all supplied by Bioswamp), cleaved caspase-3, phosphorylated (p)-Akt, and GAPDH (an endogenous control) (supplied by Abcam). Subsequently, the membranes were incubated with goat anti-rabbit IgG secondary antibody for 1 h.
Bicinchoninic acid assay kit
Manufactured by Solarbio
Sourced in China
The Bicinchoninic acid assay kit is a colorimetric assay used for the quantification of total protein concentration in a sample. The kit utilizes the reaction between protein and bicinchoninic acid to produce a purple-colored complex, which can be measured at 562 nm.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Merck Group (3 mentions)
Radioimmunoprecipitation assay lysis buffer, by Solarbio (3 mentions)
Ripa lysis buffer, by Solarbio (3 mentions)
Cleaved caspase 3, by Abcam (2 mentions)
Polyvinylidene fluoride membrane, by Beyotime (2 mentions)
Ab22555, by Abcam (2 mentions)
Gapdh, by Abcam (1 mentions)
Phosphorylated akt, by Abcam (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Antibody against β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Solarbio (1 mentions)
Phosphatase inhibitor cocktail, by Solarbio (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg secondary antibody, by Bioswamp (1 mentions)
Gapdh, by Bioswamp (1 mentions)
P erk1 2, by Abcam (1 mentions)
P drp1, by Abcam (1 mentions)
Ab193216, by Abcam (1 mentions)
Pab36269, by Bioswamp (1 mentions)
14 protocols using bicinchoninic acid assay kit
1
Protein Expression Analysis in B-CPAP Cells
2
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with radio-immunoprecipitation assay buffer supplemented with protease inhibitors. Protein amounts were determined by bicinchoninic acid assay kit (Solarbio, Beijing, China), and equal quantities of protein were loaded onto 10% Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transferring to polyvinylidene fluoride membranes. After blocking, the membranes were incubated with indicated antibodies. The antibody against β-actin (Santa Cruz Biotechnology, Dallas, TX, USA) was used as the loading control. Specific protein expression was detected by using the chemiluminescence reagents (Pierce, Rockford, IL, USA). The quantification of specific band density was done by Quantity One software (Bio-Rad, Hercules, CA, USA) with normalization to the β-actin signal.
3
Western Blot Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells or mice tumor tissues were collected and lysed in RIPA lysate buffer supplemented with 1% protease inhibitor cocktail and 1% phosphatase inhibitor cocktail (Solarbio, Beijing, China). Lysates were then centrifuged at 12,000 g for 30 min. Proteins were quantified using a bicinchoninic acid assay kit (Solarbio, Beijing, China). Resultant samples containing equal amounts of proteins were subjected to sodium dodecylsulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was blocked with TBST buffer containing 5% non-fat milk for 30 min and incubated with appropriate primary antibodies (1:1,000 dilution) in TBST at 4°C overnight. After washing with TBST, the membrane was incubated with horseradish peroxidase–conjugated secondary antibodies (1:2,000 dilution; Cell Signaling Technologies, Boston, MA) for 1 h at room temperature. Bound proteins were visualized using enhanced chemiluminescence and detected using ImageQuant LAS 4000 software.
4
Protein Expression Analysis in KGN Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole proteins were extracted from KGN cells using radioimmunoprecipitation assay lysis buffer (Solarbio), and the protein concentration was quantified using a bicinchoninic acid assay kit (Solarbio). In brief, 20 μg of proteins in each group was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, USA). The membranes were then blocked with 5% skim milk and cultured for 1 h with primary antibodies against TSC1 (Bioswamp, China), mTORC1 (Bioswamp, China), and phosphorylated (p)-mTORC1 (Abcam. USA), AKT (Bioswamp, China), p-AKT (Cell Signaling Technology, USA), and GAPDH (housekeeping control, Bioswamp, China), followed by 1 h of incubation with goat anti-rabbit IgG secondary antibody (Bioswamp, China).
5
Apoptosis and Mitochondrial Regulation in H9c2 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed to detect apoptosis-related, mitochondrial function-related, and extracellular signal-regulated kinase (ERK) protein expression in H9c2 cells. Total protein was extracted from H9c2 cells using radioimmunoprecipitation assay lysis buffer (Solarbio), and protein concentration was quantified using a bicinchoninic acid assay kit (Solarbio). Twenty micrograms of proteins from each group were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were incubated for 1 h with primary antibodies against dynamin-related protein 1 (DRP1), mitofusin 2 (MFN2), phosphorylation (p)-DRP1-S637, B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X (Bax), cleaved caspase-3, ERK1/2, p-ERK1/2, and β-actin (internal reference), followed by 1 h of incubation with the goat anti-rabbit IgG secondary antibody. All antibodies were purchased from Bioswamp, except for cleaved caspase-3 (Abcam, Cambridge, UK), p-ERK1/2 (Abcam), and p-DRP1 (CST).
6
Western Blot Analysis of Apoptosis and Mitochondrial Dynamics Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted from GES-1 cells using radioimmunoprecipitation assay lysis buffer (Solarbio), and protein concentration was quantified using a bicinchoninic acid assay kit (Solarbio). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes. After blocking with 5% skim milk, the membranes were cultured for 1 h with primary antibodies against BCL-2 associated X protein (BaX) (1 : 1000, Bioswamp, PAB30040), B-cell lymphoma-2 (Bcl-2) (1 : 1000, Bioswamp, PAB33482), cleaved caspase 3 (1 : 1000, Abcam, Ab2302), density-regulated protein 1 (DRP1) (1 : 1000, Bioswamp, PAB33409) phosphorylation (p)-DRP1 (1 : 1000, Abcam, ab193216), mitochondrial profusion protein Mitofusins (Mfn2) (1 : 1000, Bioswamp, PAB41825), occludin (1 : 1000, Bioswamp, PAB33418), claudin-1 (1 : 1000, Bioswamp, PAB33267), zona occludens 1(ZO-1) (1 : 1000, Bioswamp, PAB36669), phosphatidylinositol-3 kinase (PI3K) (1 : 1000, Bioswamp, PAB30084), p-PI3K (1 : 1000, Abcam, Ab182651), AKT (1 : 1000, Bioswamp, PAB34089), p-AKT (1 : 1000, Abcam, Ab38449), and GAPDH (1 : 1000, Housekeeping, Bioswamp, PAB36269), followed by 1 h of incubation with goat anti-rabbit IgG secondary antibody (1 : 20000, Bioswamp, SAB43714).
7
Western Blot Analysis of Myc Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted form cells using RIPA lysis buffer (Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. Total protein was quantified using a bicinchoninic acid assay kit (Beijing Solarbio Science & Technology Co., Ltd.) and 20 µg protein/lane was separated via 10% SDS-PAGE (100 V; 90 min). The separated proteins were subsequently transferred to a polyvinylidene fluoride membrane (80 V; 90 min) and blocked with 5% skim milk powder for 1 h at room temperature. The membranes were incubated with the following primary antibodies for 12 h at 4˚C: Anti-Myc (1:1,000; cat. no. sc-41; Santa Cruz Biotechnology, Inc.) and anti-GAPDH (1:5,000; cat. no. 5174; Cell Signaling Technology, Inc.). Following the primary antibody incubation, a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:5,000; cat. no. ab7090; Abcam) was incubated with the membrane for 1 h at room temperature. Protein bands were visualized using an ECL detection substrate (Thermo Fisher Scientific, Inc.).
8
Protein Expression Analysis in HESC and Endometriosis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein extraction from HESCs or endometriotic lesions was carried out using RIPA lysis buffer (Solarbio), and a bicinchoninic acid assay kit (Solarbio) was utilized for measuring the protein concentration. Protein samples (20 μg) were dissolved with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted onto polyvinylidene fluoride membranes (Beyotime), and blocked with 5% defatted milk. Next, the membranes were incubated overnight with anti-HIF1AN (ab92498, 1:5,000), anti-HIF1A (ab179483, 1:1,000), anti-VEGF (ab214424, 1:1,000), and anti-GAPDH (ab22555, 1:1,000) primary antibodies (all from Abcam) at 4℃, and then incubated at room temperature with the secondary antibody (ab6721, 1:2,000, Abcam) for 2 h. Protein bands were visualized with the enhanced chemiluminescence detection kit (Solarbio) and quantified with ImageJ software (NIH).
9
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Joint tissues were collected and total protein was extracted using 1%
radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc., Waltham,
MA, USA). Protein concentrations were measured using a bicinchoninic acid assay
kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Fifty
micrograms of each sample were prepared on ice, then separated by SDS-PAGE and
transferred to polyvinylidene difluoride membranes. After blocking with 5% (w/v)
skim milk for 2 hours and washing with Tris-buffered saline containing 0.1%
Tween-20, anti-GAPDH (diluted 1:1000, ab181602), anti-TLR-4 (diluted 1:500,
ab8378) and anti-NF-κB (p65) (diluted 1:500, ab16502) antibodies were added and
incubated overnight at 4°C. The membranes were washed again, then incubated with
secondary antibody for 1 hour. After a final wash, blots were developed using
chemiluminescence in a dark room and imaged using a gel imaging system (Bio-Rad,
Hercules, CA, USA).
radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc., Waltham,
MA, USA). Protein concentrations were measured using a bicinchoninic acid assay
kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Fifty
micrograms of each sample were prepared on ice, then separated by SDS-PAGE and
transferred to polyvinylidene difluoride membranes. After blocking with 5% (w/v)
skim milk for 2 hours and washing with Tris-buffered saline containing 0.1%
Tween-20, anti-GAPDH (diluted 1:1000, ab181602), anti-TLR-4 (diluted 1:500,
ab8378) and anti-NF-κB (p65) (diluted 1:500, ab16502) antibodies were added and
incubated overnight at 4°C. The membranes were washed again, then incubated with
secondary antibody for 1 hour. After a final wash, blots were developed using
chemiluminescence in a dark room and imaged using a gel imaging system (Bio-Rad,
Hercules, CA, USA).
10
Parotid Gland Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Parotid gland tissues (30 mg) were homogenized in 200 to 400 µL
Radioimmunoprecipitation assay-phenylmethanesulfonyl fluoride lysis buffer
(Solarbio, Beijing, China). The concentration of the supernatant was detected
using a bicinchoninic acid assay kit (Solarbio). The samples were boiled at
100°C for 10 minutes before electrophoresis. After electrophoresis, the proteins
were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA).
Then, the membranes were blocked using 8% nonfat milk in TBST buffer (50 mm
Tris-HCl, 100 mm NaCl, and 0.1% Tween-20, pH 7.4) for 3 hours. Then, the
membranes were immunoblotted with one of the following antibodies:
phosphorylated c-jun N-terminal kinase (p-JNK; Abcam, Cambridge, United
Kingdom), jun N-terminal kinase (JNK; Abcam), phosphorylated c-Jun (p-c-Jun;
Abcam), c-Jun (c-Jun; Abcam), PGC1-α (Abcam), NOX4 (Abcam), MDM2 (Abcam), Bax
(Abcam), Bcl-2 (Abcam), p53 (Abcam), Caspase-3 (Abcam), Collagen Ⅰ, Collagen Ⅲ,
and β-actin (Abcam). All blots were developed using the ECL Western Blotting
Detection Kit Reagent (Solarbio) and detected using a gel imaging analysis
system (Tanon 5200, Image Quant LAS 4000 mini, GE, Piscataway, NJ).
Radioimmunoprecipitation assay-phenylmethanesulfonyl fluoride lysis buffer
(Solarbio, Beijing, China). The concentration of the supernatant was detected
using a bicinchoninic acid assay kit (Solarbio). The samples were boiled at
100°C for 10 minutes before electrophoresis. After electrophoresis, the proteins
were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA).
Then, the membranes were blocked using 8% nonfat milk in TBST buffer (50 mm
Tris-HCl, 100 mm NaCl, and 0.1% Tween-20, pH 7.4) for 3 hours. Then, the
membranes were immunoblotted with one of the following antibodies:
phosphorylated c-jun N-terminal kinase (p-JNK; Abcam, Cambridge, United
Kingdom), jun N-terminal kinase (JNK; Abcam), phosphorylated c-Jun (p-c-Jun;
Abcam), c-Jun (c-Jun; Abcam), PGC1-α (Abcam), NOX4 (Abcam), MDM2 (Abcam), Bax
(Abcam), Bcl-2 (Abcam), p53 (Abcam), Caspase-3 (Abcam), Collagen Ⅰ, Collagen Ⅲ,
and β-actin (Abcam). All blots were developed using the ECL Western Blotting
Detection Kit Reagent (Solarbio) and detected using a gel imaging analysis
system (Tanon 5200, Image Quant LAS 4000 mini, GE, Piscataway, NJ).
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!