Whole cells were harvested in total protein extraction buffer (TPEB; Transgen Biotech, China) containing a protease inhibitor cocktail (Roche, Germany), and the protein concentration was measured by the BCA protein assay (Cwbiotech, China). Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were incubated with various primary antibodies, followed by a horseradish peroxidase (HRP)-conjugated secondary antibody. The signals were detected using an Immobilon Western Chemiluminescent HRP Substrate kit (Thermo Fisher, USA). The quantification of Western blot analysis was performed by using Quantity One software, and the protein expression levels were normalized to β-actin levels.
Bca protein assay
Manufactured by CWBIO
Sourced in China
The BCA protein assay is a colorimetric-based method used for the quantitative determination of total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reaction to detect and quantify the presence of protein in a solution.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Phosphatase inhibitor, by Applygen (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Immobilon western chemiluminescent hrp substrate kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Chemiluminescence detection reagent, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Af0006, by Beyotime (1 mentions)
Af0003, by Beyotime (1 mentions)
Sc 73632, by Santa Cruz Biotechnology (1 mentions)
Nbp1 45525, by Novus Biologicals (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
Protease inhibitor cocktail, by CWBIO (1 mentions)
Glut4, by Cell Signaling Technology (1 mentions)
Odyssey clx infrared imaging system, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit secondary antibody, by LI COR (1 mentions)
11 protocols using bca protein assay
1
Protein Expression Analysis by Western Blot
2
Quantifying Dental Stem Cell Protein
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The total protein of hDPSCs was collected and a BCA protein assay (Cwbio, Cambridge, MA, USA) was performed to measure the concentration. An amount of 20 μg of proteins was separated on 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked and incubated overnight at 4 °C with the following primary antibodies: FBXO32 (1:1000; 67172-1-Ig, Proteintech, Wuhan, China), anti-DSPP antibody (1:1000; sc-73632, Santa-Cruz Biotechnology, Santa Cruz, CA, USA), anti-DMP1 antibody (1:1000; NBP 1-45525, Novus Biologicals, Centennial, CO, USA), anti-GAPDH antibody (1:1000; AF0006, Beyotime, Shanghai, China) and anti-β-actin antibody (1:1000; AF0003, Beyotime). Subsequently, the membranes were incubated with secondary antibodies (1:2000, Beyotime). The immunoreactive bands were detected using chemiluminescence detection reagents (Millipore) and visualized by a detection system (Bio-Rad, Hercules, CA, USA). The intensities of each sample were quantified using ImageJ software.
3
Protein Extraction and Western Blotting Protocol
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Protein was extracted from cells using RIPA buffer containing PMSF (Beyotime Biotechnology, China). Total protein levels were quantified using the BCA protein assay (CWBio, China). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were blocked in 1% BSA before the incubation with various primary antibodies (Table 1 ) overnight at 4°C. Secondary antibody incubations were carried out at room temperature for 1 h. Immune complexes were detected and evaluated using the SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, USA) and the band density was quantified with Image J (NIH, USA).
4
Quantitative Western Blot Analysis of Apoptosis Proteins
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Ovary tissues from different groups were homogenized with 200 μL RIPA lysis buffer mixed with 1× protease inhibitor Cocktail (CWBiotech), and the homogenates were incubated on ice for 20 min. After centrifugation at 10 000 × g for 10 min, the supernatants were obtained and total tissue protein concentrations were measured by the BCA protein assay (CWBIO, China). Protein samples were mixed with a 5× SDS-PAGE sample loading buffer and boiled for 5 min. Equal amounts of protein (20 μg) were electrophoresed through 12% SDS-PAGE and then transferred onto a polyvinylidene fluoride (PVDF) membrane, which was activated in methanol for 1 min. The PVDF membranes were blocked with nonfat milk at room temperature for 2 h and then washed three times (10 min each) with Tris-buffered saline (TBS) containing 0.05% Tween-20 (TBST). The PVDF membranes were incubated with anti-Bax and anti-Bcl-2 antibodies (1 : 200 dilution, Santa Cruz Biotechnology, USA) at room temperature for 2 h and washed three times (10 min each) with TBST. The membranes were incubated with species-matched horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. After washing three times with TBST, the specific protein signals were visualized using an ECL Plus (Thermo Fisher Scientific). The results of western blot were quantified using the ImageJ software.
5
Protein Expression Analysis by Western Blot
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The cells were washed with PBS and then lysed on ice in a radioimmunoprecipitation assay (RIPA) buffer (Kangwei, Beijing, China). The total protein concentration was measured by bicinchoninic acid (BCA) protein assay (CWBIO, Beijing, China). Subsequently, 30 μg of protein was subjected to SDS-PAGE and subsequently transferred to PVDF membranes (Millipore Corporation, Billerica, MA, United States). The membranes were blocked for 1 h with 5% non-fat milk before being incubated with primary antibodies [HK2, PKM2, GLUT4: Cell Signaling Technology, Danvers, USA. LC3-I/II: Sigma-Aldrich, Germany; ATG5, Beclin1, PFKM, β-actin: Proteintech Group, Inc., Chicago, IL, USA; monoclonal anti-enterovirus antibody (viral capsid protein, VP1) Dako, Carpinteria, CA, USA] at 4°C overnight, followed by incubation with an IRDye®800CW goat anti-rabbit secondary antibody (1:8,000, 926-32211, LI-COR®, USA) for another 1 h. Immunoblotting bands were visualized under an Odyssey CLx infrared imaging system (LI-COR®, USA). Protein expression levels were quantified by densitometry using the ImageJ software and were normalized to β-actin.
6
Western Blot Analysis of PAI-1 Expression
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Lung tissues were dissected and lysed in radio-immunoprecipitation assay lysis buffer (Beyotime, Shanghai, China). BCA protein assay (CWBIO, Beijing, China) was used to quantify the amount of protein. Equal quantities of protein were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, CWBIO, Beijing, China) and transferred to PVDF membranes. The membranes were blocked for 1 h using a blocking solution. Samples were incubated overnight with anti-PAI-1 (1:1000, no. ab66705, Abcam) and anti-GAPDH (1:2500, no. AF1186, Beyotime, Shanghai, China). The membrane was washed twice with TBS, and bound antibody was detected using IgG-HRP secondary antibodies, HRP-conjugated goat anti-rabbit IgG and HRP-conjugated goat anti-mouse IgG (no. A0208 and no. A0216, Beyotime, Shanghai, China), at a ratio of 1:1000 for 2 h at room temperature. Finally, membranes were stained with ECL reagent (Beyotime, Shanghai, China), scanned, and stored using a gel imaging system. Optical density of the immunoreactivity was measured and analyzed with an Alpha Imager.
7
Tissue Protein Extraction and Analysis
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The total protein extraction from tissues or cells was conducted with radioimmunoprecipitation assay (RIPA) lysis buffer (P0013c, Beyotime Biotechnology, Shanghai, China) containing phenyhnethylsulfonyl fluoride (PMSF). The supernatant was incubated on ice for 30 min, and centrifuged at 4 °C and 8000 g for 10 min, followed by bicinchoninic acid (BCA) protein assay (CWBio, Beijing, China). The extracted proteins were stored at – 80 ℃ after packaging. The proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h, transferred onto nitrocellulose membranes, and then blocked with 5% skimmed milk (PBS) for 2 h. The membranes were incubated for 30 min at ambient temperature with rabbit polyclonal antibodies MMP11 (1:1000, ab119284), Bcl-2 (1:1000, ab182858), Cyclin D1 (1:1000, ab40754), CDK4 (1:1000, ab199728), Bax (1:1000, ab182733), and GAPDH (ab9485, 1:2500) (Abcam, Cambridge, UK). HRP-coupled antibody to immunoglobulin G (IgG) (A21020, 1:5000, AmyJet Scientific Inc., Wuhan, China) was loaded for 1-h incubation. The immunoblots were subsequently developed using enhanced chemiluminescence reagent (BB-3501, Amersham Biosciences, Piscataway, NJ), and imaged on the gel imager. An imaging system (Bio-Rad Inc., Hercules, CA) was utilized for gray value analysis with the application of Quantity One v4.6.2 software [24 (link)].
8
Protein Expression Analysis by Western Blot
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Cell lysates were harvested using RIPA buffer, according to the manufacturer's instructions (CWBIO, Beijing, China). Protein concentration was determined using the BCA protein assay according to the manufacturer's instructions (CWBIO, Beijing, China). p-ERK1/2, ERK1/2, p-AKT, AKT, p-NF-κB, NF-κB, Bcl-2, Bax, Cyt C, cleaved caspase-9, and cleaved caspase-3 proteins were detected using Western blot, as described previously [39 (link)]. In brief, samples were separated in 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. After 1 h incubation in 5% (w/v) milk powder in PBS, the membranes were incubated with primary antibodies overnight, followed by incubation with secondary antibodies. Proteins were detected using WesternBright™ ECL (Advansta, California, USA) with a gel imaging analysis system (Tanon, Shanghai, China).
9
Protein Expression Analysis in Dental Cells
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The protein lysis buffer containing a phosphatase inhibitor (Applygen Technologies Inc., Beijing, China) was used to harvested cells. The cell suspensions were centrifuged at 4°C for 30 min with a speed of 12,000 × g. The BCA Protein Assay (CWBIO, Beijing, China) was used to determine the protein concentration, and each lane was loaded with equal aliquots of the total protein (20 μg). The sample lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA), blocked in blocking sodium (Beyotime, Shanghai, China) for 1 h, and probed with the following antibodies at 4°C overnight: DSPP (1 : 1000; Santa Cruz Technology, Santa Cruz, CA), DMP1 (1 : 1000; Bioss, Beijing, China), p38, p-p38, and β-actin (1 : 10000; Cell Signaling Technology, Beverly, MA, USA). The membrane was incubated at room temperature for 1 h with horseradish peroxidase- (HRP-) conjugated antirabbit immunoglobulin. The protein expression was detected using a Western enhanced chemiluminescence blotting kit (ECL, SOLIBRO, Beijing, China).
10
Quantifying Protein Expression in Mammalian Cells
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The cells were harvested with protein lysis buffer containing a phosphatase inhibitor (Applygen Technologies Inc., Beijing, China). The cell suspensions were centrifuged at 4 °C for 30 min at 12,000 × g. The protein concentration was determined using bicinchoninic acid (BCA) Protein Assay (CWbio, Beijing, China) and each lane was loaded with equal aliquots of total protein (20 μg). The lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA), blocked in 5% bovine serum albumin for 2 h, and probed with antibodies against the following at 4 °C overnight: IGFBP7 (sc-365293, 1:1000, Santa Cruz Biotechnology, CA, USA), DSPP (sc-73632, 1:1000, Santa Cruz Biotechnology, CA, USA), and β-actin (#4970, 1:10,000, Cell Signaling Technology, Beverly, MA, USA). The membrane was incubated at room temperature for 1 h with horseradish peroxidase-conjugated anti-rabbit immunoglobulin. Protein expression was detected using a western enhanced chemiluminescence blotting kit (Solibro, Beijing, China).
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