Cells were collected, washed with PBS, and then lysed with lysis buffer (150 mM NaCl, 1% NP-40, 0.5% deoxycholate, 0.1% SDS, 50 mM Tris (pH 8.0, and 5 mM EDTA) containing protease inhibitors (10 μg/mL phenylmethylsulfonyl fluoride). After quantification with BCA assay (Beyotime Biotechnology, Beijing, China), 30 μg total proteins were loaded and separated on 10% SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked with 5% non-fat milk, probed with rabbit polyclonal antibodies against human LC3 or cleaved LC3 (LC3-II) or Beclin 1 (Cell Signaling Technology, USA), mouse monoclonal antibody against human β-actin (Cell Signaling Technology, USA), and ASPP2 (sigma, Germany). The membranes were washed, probed with HRP conjugated secondary antibody, and detected with chemiluminescent kit. The western blot images were quantitatively analyzed with Image J software (National Institutes of Health, USA).
Aspp2
Manufactured by Merck Group
Sourced in United States
ASPP2 is a laboratory equipment product manufactured by Merck Group. It is a high-performance instrument designed for specific applications in scientific research and analysis. The core function of ASPP2 is to enable precise and efficient data collection and processing, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Beclin 1, by Cell Signaling Technology (2 mentions)
Lc3 2, by Cell Signaling Technology (1 mentions)
Mouse monoclonal antibody against human β actin, by Cell Signaling Technology (1 mentions)
Bca assay, by Beyotime (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Cyclin b1, by Cell Signaling Technology (1 mentions)
Phospho p70s6k t389, by Cell Signaling Technology (1 mentions)
Cyclin a2, by Cell Signaling Technology (1 mentions)
Phospho 4ebp1 t37 46, by Cell Signaling Technology (1 mentions)
Phospho s6 s235 236, by Cell Signaling Technology (1 mentions)
Goat anti rabbit secondary antibody conjugated with horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Cyclin e1, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Vimentin, by Santa Cruz Biotechnology (1 mentions)
Image studio system ecl, by LI COR (1 mentions)
Gapdh, by Merck Group (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Hif 1α, by Abcam (1 mentions)
4 protocols using aspp2
1
Western Blot Analysis of Autophagy Markers
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
These cells were lysed on ice for 30 min with the assistance of RIPA buffer containing protease inhibitor. Protein was detected by SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated using the appropriate primary antibody (ASPP2, LC3B from Sigma and caspase3, cleaved caspase3, Glucose-Regulated Protein 78 (GRP78), Chop, p62 (sequestosome 1), phospho-mTOR, phospho-S6 (Ser235/236), phospho-p70S6K (Thr389) and β-actin antibody from cell signaling technology) at 4°C. After being washed for three times, the membrane was incubated with the corresponding secondary antibody (cell signaling technology) for 1 h at room temperature. Finally, the membrane was photographed using a gel imaging system. Moreover, optical density analysis was conducted using ImageJ software, with the relative levels of proteins in each group normalized to a loading control.
3
Western Blot Analysis of Autophagy Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissues were lysed with a homogenizer in RIPA buffer with protease inhibitors. Total proteins were extracted using centrifugation and denatured via boiling. Then, 50 μg of protein were loaded onto a 12% separation gel and a 5% concentration gel. The total proteins were separated into many bands by electrophoresis and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, CA, USA) by electroblotting. The membranes were incubated with the rabbit primary antibodies against ASPP2, LC3B (Sigma, MO, USA), p62, Atg7, Beclin-1, CyclinA2, CyclinB1, CyclinE1, phospho-4EBP1 T37/46, phospho-S6 S235/236, and phospho-P70S6K T389 (Cell Signaling, CA, USA). Then, the membranes were incubated with a goat anti-rabbit secondary antibody conjugated with horseradish peroxidase (Cell Signaling, CA, USA). Finally, immunoreactive bands were developed using a chemiluminescent substrate (Thermo Fisher Scientific, IL, USA). The grayscale was analyzed by ImageJ software, and the relative grayscale value was normalized to that of β-actin.
4
Proteomic Analysis of Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from cells using urea buffer (2 M Thiourea, 4%CHAPS, 40 mM Tris-Base, 40 mM DTT, 2%Pharmalyte). Equal amount of proteins were separated at sodium dodecyl sulfate-polycylamide gel electrophoresis (SDS-PAGE), and then transferred to PVDF membranes using the cold transfer buffer. Membranes were blocked with 5% non-fat milk in TBS-T for 1 h at room temperature, and subjected to corresponding primary antibody at 4 °C overnight. Antibodies used were list as bellow: ASPP2 (Sigma-Aldrich, St Louis, MO, USA, 1 : 2000), β-actin (Sigma-Aldrich, 1 : 2000), HIF-1α (Abcam, Cambridge, UK, 1 : 750), Vimentin (Santa Cruz, CA, USA, 1 : 2000), E-cadherin (Santa Cruz, 1 : 2000) and GAPDH (Sigma-Aldrich, 1 : 2000). Secondary antibodies, goat anti-mouse-HRP and goat anti-rabbit-HRP, were diluted at 1 : 2000. Signals were visualized by ECL. Membrane was then ready for scanning by Image studio system (ECL, LI-COR, Lincoln, Georgia, USA). Protein quantification was conducted by Image J software (National Institutes of Health, Bethesda, MD, USA). The gray values of protein were achieved as gray level of protein band/gray level of loading control.
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!