Cells were lysed in RIPA buffer (PMSF = 1 mmol/l, Solarbio), and proteins were isolated. Then protein concentration was measured using a BCA protein concentration Assay Kit (Beyotime). The proteins were separated by 10 % SDS-PAGE (20 μg protein in each lane, Solarbio) and transferred to PVDF membranes (Millipore, Billerica, MA, USA). OSTF1 (1:1000, 10671-1-AP, Proteintech, Wuhan, China), cl-caspase 3 (1:1000, AF7022, Affinity, Changzhou, China), cl-PARP (1:1000, AF7023, Affinity), aggrecan (1:1000, DF7561, Affinity), collagen-II (1:1000, AF0135, Affinity), MMP1 (1:1000, A22080, Abclonal, Wuhan, China), MMP13 (1:1000, AF5355, Affinity), p-p65 (1:1000, AF2006, Affinity) and p65 (1:1000, AF5006, Affinity) primary antibodies were added and incubated overnight at 4 °C, followed by incubation with the respective secondary antibodies (1:3000) at 37 °C for 1 h. ECL reagent (Solarbio) was used for luminescence detection, and data quantification was utilized by Gel-Pro-Analyzer software.
Af7023
Manufactured by Affinity Biosciences
Sourced in United States
The AF7023 is a laboratory instrument designed for cell culture applications. It functions as an incubator, providing a controlled environment for the growth and maintenance of cell cultures. The AF7023 regulates temperature, humidity, and CO2 levels to create optimal conditions for cell proliferation and differentiation.
Automatically generated - may contain errors
Lab products found in correlation
Af7022, by Affinity Biosciences (4 mentions)
Af0135, by Affinity Biosciences (1 mentions)
Af2006, by Affinity Biosciences (1 mentions)
Af5355, by Affinity Biosciences (1 mentions)
Ecl reagent, by Solarbio (1 mentions)
Df7561, by Affinity Biosciences (1 mentions)
Af5006, by Affinity Biosciences (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Bca protein concentration assay kit, by Beyotime (1 mentions)
Bs 5092r, by Bio-Synthesis (1 mentions)
Ripa lysate, by Solarbio (1 mentions)
Af6139, by Affinity Biosciences (1 mentions)
Bca assay kit, by Solarbio (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab191217, by Abcam (1 mentions)
Af0131, by Affinity Biosciences (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Af4039, by Affinity Biosciences (1 mentions)
4 protocols using af7023
1
Western Blot Analysis of Osteoarthritis Markers
2
Liver and Macrophage Protein Detection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
TL1A (bs-5092R, Biosynthesis, China), CCR2 (DF7507, Affinity, USA), CXCR2 (DF7095, Affinity, USA), cleaved-caspase3 (AF7022, Affinity, USA), and cleaved-PARP1 (AF7023, Affinity, USA) were detected. The extraction of total protein was from liver tissues and macrophages. Equal amounts of total proteins were fractionated by SDS-PAGE, transferred onto polyvinylidene difluoride membranes, and then incubated with corresponding primary antibodies overnight at 4°C. Then, the membranes incubated with the secondary antibody, respectively, at 37°C for 1 hour.
3
Western Blot Analysis of Spinal Cord Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For western blot assay, total proteins were extracted from spinal cord tissues and homogenized in RIPA lysates (Solarbio, Beijing, China) supplemented with phenylmethyl sulphonyl fluoride (PMSF). The protein concentration was evaluated by the BCA assay kit (Solarbio). Proteins were resolved on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, transferred to the polyvinylidene fluoride membranes (MilliporeSigma, St. Louis, USA), and blocked in the non-fat powdered milk. The membranes were then incubated with primary antibody at 4°C overnight. After incubation with HRP-conjugated secondary antibodies, the blots were developed with enhanced chemiluminescence substrate reagents (Solarbio). Primary antibodies used in the present study included HGFIN (1:5000; Proteintech Genomics, San Diego, USA; 66926-1-Ig), cleaved PARP (1:1000, AF7023; Affinity Biosciences, Cincinnati, USA), cleaved caspase-3 (1:1000, AF7022; Affinity), Bcl-2 (1:1000, AF6139; Affinity), AKT (1:3000, 10176-2-AP; ProteinTech), and p-AKT (1:2000, 66444-1-Ig; Protein Tech).
4
Protein Expression Analysis by Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted using Radio Immunoprecipitation Assay (RIPA) buffer (P0013B, Beyotime, Jiangsu, China) containing 1 × protease inhibitor cocktail [36 (link)]. Total protein was separated by 12% SDS-PAGE and transferred onto polyvinylidene fluoride membranes (IPFL00010, Merck, MA, USA). The bands were incubated overnight with primary antibodies at 4°C. Primary antibodies are listed below: cleaved caspase 3 (AF7022, Affinity, CA, USA; 1:1000), caspase 3 (AF6311, Affinity; 1:1000), cleaved poly ADP-ribose polymerase (PARP; AF7023, Affinity; 1:1000), PARP (ab191217, Abcam, Cambridge, UK; 1:1000), cyclin D1 (AF0931, Affinity; 1:1000), cyclin dependant kinase 2 (CDK2; AF6237, Affinity; 1:1000), p27 (AF6324, Affinity; 1:1000), epithelial €-cadherin (AF0131, Affinity; 1:1000), neural (N)-cadherin (AF4039, Affinity; 1:1000), IGF1R (AF6125, Affinity; 1:1000), p-PI3K (AF3241, Affinity; 1:1000), p-AKT (ab38449, Abcam; 1:1000), p-mTOR (AF3308, Affinity; 1:1000), and GAPDH (ab9485, Abcam; 1:1000). The following day, the bands were incubated with goat anti-rabbit IgG (H + L) HRP (S0001, Affinity; 1:5000) for 1 h at room temperature. The results were visualized with ECL reagent (WBKlS0010, Merck), photographed with Gel Imager System, and analyzed using Image J software (1.8.0, National Institutes of Health, USA).
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!