Total protein was extracted using RIPA lysate with Protease Inhibitor Cocktail (Roche, Germany), and protein concentrations were measured using a bicinchoninic acid protein assay (Beyotime). Proteins (20 μg) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Bio-Rad, USA) and transferred onto a polyvinylidene fluoride (PVDF) membrane (EMD Millipore, USA). After blocking in QuickBlock Blocking Buffer (Beyotime), the membrane was cut according to the molecular weight of the prestained marker protein and incubated overnight at 4°C with primary antibodies. Immune complexes were incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies at a concentration of 1 : 6000 (Yeasen, China). Enhanced chemiluminescence reagents (EMD Millipore) were added to visualize protein bands, and images were captured using a ChemiDoc MP system (Bio-Rad). ImageJ software (National Institutes of Health, Bethesda, MD) was used to calculate the intensity (gray value) of each protein band to enable comparison. Primary antibodies were purchased from the following commercial sources: collagen type I alpha 1 (COL1A1), bone sialoprotein (BSP) (1 : 1000; CST, USA), DSPP, osteocalcin (OCN) (1; 200; Santa Cruz Biotechnology, USA), DMP-1 (1 : 1000; Novus, USA), and glyceraldenhyde-3-phosphate dehydrogenase (GAPDH) (1 : 10000; Proteintech, USA).
Bicinchoninic acid protein assay
Manufactured by Beyotime
Sourced in China
The Bicinchoninic acid (BCA) protein assay is a colorimetric method used to quantify the total protein content in a sample. The assay is based on the reduction of Cu2+ to Cu+ by proteins in an alkaline medium, followed by the chelation of the Cu+ ions with bicinchoninic acid, resulting in a purple-colored complex that can be measured spectrophotometrically. This method provides a simple, sensitive, and stable way to determine protein concentrations across a wide range of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (10 mentions)
Pvdf membrane, by Merck Group (7 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (4 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Polyvinylidene difluoride membrane, by Merck Group (4 mentions)
Nitrocellulose membrane, by Merck Group (3 mentions)
Gapdh, by Abcam (3 mentions)
Lysis buffer, by Beyotime (3 mentions)
Phenylmethylsulfonyl fluoride, by Beyotime (3 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Protease inhibitor, by Beyotime (2 mentions)
Ab6721, by Abcam (2 mentions)
Beclin 1, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Proteintech (2 mentions)
Alp assay kit, by Nanjing Jiancheng (2 mentions)
Ecl detection system, by Cytiva (2 mentions)
Quantity one 4, by Bio-Rad (2 mentions)
Nitrocellulose membrane, by Beyotime (2 mentions)
Gapdh, by Boster Bio (2 mentions)
Proteinase inhibitor cocktail solution, by Roche (2 mentions)
60 protocols using bicinchoninic acid protein assay
1
Protein Extraction and Western Blot Analysis of Bone Markers
2
Serum and Kidney Sample Collection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After 12 weeks of the experimentation, mice were anesthetized with pentobarbital and exsanguinated by cardiac puncture. Blood samples were collected and centrifuged at 3000g for 15 min at 4 °C; then, the supernatants (serum) were collected and stored at − 80 °C for future analysis. Kidneys were either frozen in liquid nitrogen then transferred to − 80 °C refrigerator for further analysis or fixed in buffered 10% formalin for histological examination and immunohistochemistry. Kidney tissues were homogenized by RIPA lysis buffer (Beyotime, Beijing, China), then centrifuged at − 4 °C. After the supernatants were extracted, the protein concentrations were detected with bicinchoninic acid protein assay (Beyotime, Beijing, China); then, the supernatants were preserved in − 80 °C refrigerator.
3
Western Blot Analysis of Protein Biomarkers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with 10 μL/mL Radio-Immunoprecipitation assay Lysis Buffer containing protease inhibitors (Beyotime), centrifuged at 14,000 × g, and quantified by bicinchoninic acid protein assay (Beyotime). Total protein (20 μg) was loaded into a sodium lauryl sulfate polyacrylamide gel and separated. Then, the sample was electroblotted onto a polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), blocked with 5% skim milk, and incubated with the following primary antibodies: β-actin (A5441, MilliporeSigma), KLF6 (14716-1-AP, Proteintech), Nrf2 (16396-1-AP, Proteintech), NF-κB (14220-1-AP, Proteintech), p-NF-κB (8242, Cell Signaling Technology), HO-1 (10701-1-AP, Proteintech), Bax (50599-2-Ig, Proteintech), Bcl-2 (12789-1-AP, Proteintech), and Ki-67 (27309-1-AP, Proteintech). The membrane was then incubated with the secondary antibody conjugated with horseradish peroxidase (Beyotime), developed by enhanced chemiluminescence kit (Amersham Pharmacia Biotech, Little Chalfont, UK) and imaged by ChemiDoc XRS imaging system. Image J image analysis software was used for data analysis. Three independent replicates were performed for each group.
4
Myocardial Protein Expression Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A total of 3 days after surgery, tissue samples of the left ventricular myocardium were homogenized in in RIPA lysis buffer containing 1% PMSF. Protein concentrations in supernatants were measured with a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). Equal amounts of prepared proteins (50 µg/lane) were subjected to 10% SDS-PAGE, separated by electrophoresis and transferred to nitrocellulose membranes. Following blocking in 5% non-fat milk PBS for 2 h, the membranes were incubated overnight at 4°C with anti-Nox2 (cat. no. ab80508; Abcam; 1:1,000), anti-Nox4 (cat. no. ab195524; Abcam; 1:1,000), anti-Bax (cat. no. ab32503; Abcam; 1:1,000), anti-Bcl-2 (cat. no. ab182858; Abcam; 1:1,000) or β-actin (cat. no. ab8227; Abcam; 1:1,000) primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (cat. no. 7074; Cell Signaling Technology, Inc.; 1:5,000) for 1 h at room temperature. Immunoreactive bands were detected using an enhanced chemiluminescence system (EMD Millipore) and quantified by Image-Pro Plus v.6.0.
5
Western Blot Analysis of Immune Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as described before6 (link). The whole cell extracts were prepared with lysis buffer. The protein concentrations were determined using a bicinchoninic acid protein assay (Beyotime Institute of Biotechnology). Protein samples (50 μg/lane) were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% resolving gels and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked with 5% non-fat milk for 1 h at room temperature and then incubated with the following primary antibodies overnight at 4 ℃: PD-1 (1:1000), p-Stat5 (1:1000), Stat5 (1:1000), cleaved Caspase 3 (1:1000), CD4 (1:1000), CD8 (1:1000), and Tubulin (1:1000). All antibodies were purchased from Cell Signaling Technology, Inc. Horseradish peroxidase-conjugated anti-rabbit or anti-mouse immunoglobulin G secondary antibodies (1:2000; Cell Signaling Technology, Inc.) were used for 1 h at room temperature. Specific immune complexes were visualized using enhanced chemiluminescence (Beyotime Institute of Biotechnology). The results of western blot were semi-quantified with Quantity One software (Version 4.62; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
6
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were collected in phosphate buffered solution (PBS) and washed twice with cold PBS by centrifuging at 300 × g for 3 min. All procedures were carried out at 4°C unless otherwise mentioned. Total proteins were extracted using RIPA lysis buffer (Beyotime Biotechnology). Nuclear and cytoplasmic extraction kit from KeyGEN BioTECH Corp.Ltd., was used to harvest cytosolic and nuclear proteins, according to the manufacturer's instructions. Then, bicinchoninic acid protein assay (Beyotime Biotechnology) was applied to measure protein concentrations. Equal amounts of prepared proteins were loaded on 10%–12% sodium dodecyl sulfate polyacrylamide gels. Subsequently, proteins were transferred onto polyvinylidene difluoride membranes (Merck Millipore), which were then blocked in 5% skim milk for 2 h. Then membranes were probed with primary antibodies including PARP (1:1000), GSTpi (1:1000), FLAG (1:1000), GAPDH (1:1000), β‐actin (1:1000) and Lamin B2 (1:1000). After incubation overnight, IRdye 680‐labeled IgG secondary antibodies (1:5000) were incubated for 1 h. The Odyssey infrared imaging system (LI‐COR Biosciences) was applied for protiens visualization. β‐actin, GAPDH and Lamin B2 were utilized as internal controls, respectively.
7
Protein Expression Analysis in Testis and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins isolated from testes of the mice and the MLTC-1 cells were obtained following the standard procedures and quantified by using the bicinchoninic acid protein assay (Beyotime, Shanghai, China). The proteins were separated by 12% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore) and incubated with the rabbit anti-VEGFA (ab46154, 1 : 1000, Abcam, Cambridge, USA), rabbit anti-VEGFR1 (ab32152, 1 : 100, Abcam, Cambridge, USA), rabbit anti-VEGFR2 (ab5473, 1 : 100, Abcam, Cambridge, USA), rabbit anti-eNOS (ab5589, 1 : 100, Abcam, Cambridge, USA), and rabbit anti-GAPDH (ab9485, 1 : 2500, Abcam, Cambridge, USA) overnight at 4°C. On day 2, after washing with TBS three times, the membranes were incubated with goat anti-rabbit HRP-conjugated secondary antibodies (ab6721, 1 : 5000, Abcam, Cambridge, USA) at 37°C for 1 h. The relative protein levels in each sample were normalized to the levels of GAPDH to standardize the variations.
8
Western Blot Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples were lysed in RIPA buffer supplemented with protease inhibitors and subjected to ultrasonics at low frequency. The supernatant containing total proteins was harvested after centrifugation and the protein concentration was measured by bicinchoninic acid protein assay (Beyotime, China). Equal amounts (30 μg) of total proteins were loaded on 10% or 15% SDS-PAGE according to the molecular weight of the target proteins. Proteins were transferred onto polyvinylidene fluoride membranes (Millipore), and blocked in 5% BSA for 2 h at room temperature, followed by incubation overnight at 4°C with primary antibodies for LC3 (Cell Signaling Technology, USA), Beclin1 (Cell Signaling Technology, USA), ATG5 (Cell Signaling Technology, USA), P62 (Proteintech, USA), ALP (R&D systems, USA), Runx2 (Cell Signaling Technology, USA), PPAR-γ (Abcam, UK), OPG (Abcam, UK), RANKL (Abcam, UK) and Actin (Abcam, UK). After rinsed with tris buffered saline supplemented with tween (TBST) three times, membranes were incubated with secondary antibodies (Cowin Biotech Co, Beijing, China) for 1 h at room temperature. Blots were captured with a Western-Light Chemiluminescent Detection System (Tanon, Shanghai, China).
9
Protein Expression Analysis of NPCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NPCs lysates were prepared in RIPA buffer (Thermo) and then extracted protein. Bicinchoninic acid protein assay (Beyotime) was applied for detecting concentrations. Protein was subjected to SDS-PAGE and transferred to a PVDF membrane for immunoblotting analysis. The membranes were incubated with anti-PRKCA (CST, 2056, 1:1000), anti-COL2A1 (Proteintech, 28,459–1-AP, 1:1000) and anti-GAPDH (Proteintech, 60,004–1-Lg, 1:20,000) overnight at 4 °C, and then incubated with goat anti-rabbit IgG-HRP (abcam, ab6721, 1/20000) and goat anti-mouse IgG-HRP (abcam, ab205719, 1:1000) at 24 °C for 2 h. Finally, membranes were exposed to a chemiluminescence imaging analysis and quantified with Image J software.
10
Western Blotting for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed following a standard method that has been previously reported [25 (link)]. Briefly, the protein concentration was determined using the bicinchoninic acid protein assay (Beyotime, Shanghai, China). The same amount of protein in the total cell extract was isolated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane, which was then blocked with 5% (wt/vol) fat-free milk. The membrane was continuously incubated with specific primary antibodies and their respective secondary antibodies, with β-actin or GADPH as a loading control. The immunoreactive bands were detected by chemiluminescent reagents. The results were scanned by BandScan to analyze the grayscale values of the film.
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!