Tissues or cells were lysed with protein lysate buffer radio immunoprecipitation assay (RIPA) (Beyotime, P00138). Supernatant of lysate was collected, and the protein concentrations were detected by bicinchoninic acid (BCA) method (Beyotime, P0012). The protein was isolated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was sealed with 5% skim milk solution at room temperature for 30 min and then incubated overnight at 4 °C with corresponding primary antibody, including Bcl-2 rabbit polyclonal antibody (Beyotime, AF0060, 1:500), Bax rabbit monoclonal antibody (Beyotime, AF1270, 1:500), LC3 rabbit polyclonal antibody (protein technology, 18,725–1-AP, 1:500), Pink1 rabbit polyclonal antibody (protein technology, 23,274–1-AP, 1:500), Parkin rabbit polyclonal antibody. The next day, the membrane was washed with tris buffered saline tween (TBST) buffer and incubated with diluted horseradish peroxidase labeled goat anti-rabbit antibody (Beyotime, A0208, 1:1000) and anti-mouse antibody (Beyotime, A0216, 1:1000) at room temperature for 1 h. The ultra-sensitive chemiluminescence kit BeyoECL Star was used for color development, and the results were analyzed with image J 8.0.
Af1270
Manufactured by Beyotime
Sourced in China, United States
The AF1270 is a laboratory equipment product. It serves a core function in the scientific research and testing environment.
Automatically generated - may contain errors
Lab products found in correlation
Af0060, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
A0216, by Beyotime (1 mentions)
P00138, by Beyotime (1 mentions)
A0208, by Beyotime (1 mentions)
P0012, by Beyotime (1 mentions)
Protein lysis solution, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Af1186, by Beyotime (1 mentions)
Bca protein concentration determination kit, by Beyotime (1 mentions)
Af1681, by Beyotime (1 mentions)
Af7209, by Beyotime (1 mentions)
Af2155, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Chemiluminescence imaging system, by Bio-Rad (1 mentions)
Ab129002, by Abcam (1 mentions)
Ab207298, by Abcam (1 mentions)
Ab181593, by Abcam (1 mentions)
Ab189139, by Abcam (1 mentions)
4 protocols using af1270
1
Western Blot Analysis of Apoptosis and Mitophagy Markers
2
Protein Extraction and Western Blot Analysis
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Total protein was extracted using protein lysis solution (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Then Bicinchoninic Acid (BCA) protein concentration determination kit (Beyotime, Shanghai, China) was used to quantify the extracted protein. After adding sodium dodecyl sulfate (SDS)-loading buffer, it was placed in boiling water at 98°C for 5 min to denature. The sample was separated by 12% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE). Then the protein was transferred to polyvinylidene fluoride (PVDF, Millipore, Bedford, USA) membrane by Western electrophoresis. The membrane was incubated in 5% skimmed dry milk for 2 h. After incubation, they were incubated overnight with primary antibody: Bcl-2 (1RV 1000, AF0060, Beyotime, Shanghai, China), Bax (1RV 1000, AF1270, Beyotime, Shanghai, China), caspase1 (1RV 1000, AF1681, Beyotime, Shanghai, China), IL-1 β (1RV 1000, AF7209, Beyotime, Shanghai, China), NLRP3 (1RV 1000, AF2155, Beyotime, Shanghai, China) and GAPDH (1RV 1000, AF1186, Beyotime, Shanghai, China). The corresponding second antibody was horseradish peroxidase labeled goat anti-rabbit IgG (H+L) (A0208,1VO1000, Beyotime, Shanghai, China). Finally, the PVDF films were treated with Clarity Western ECL substrate (1705061 USA) and observed by chemiluminescence imaging system (Bio-Rad, USA).
3
Western Blot Analysis of Cell Signaling Proteins
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Western blot analysis was performed as described previously27 (link). Cells were grown to 80%–90% confluency in 6-well plates as described above and lysed in RIPA lysis buffer supplemented with 1 mmol/L PMSF. The proteins were separated by SDS-PAGE, transferred onto PVDF membranes and then incubated with the appropriate primary antibodies. Primary antibodies against Cyclin B1 (ab181593, Abcam), CDK1 (ab1333327, Abcam), KIF20A (ab70791, Abcam), PLK1 (ab189139, Abcam), FOXM1 (ab207298, Abcam), GRP78 (3177T, Cell Signaling), Bax (AF1270, Beyotime), caspase-3 (AF0081, Beyotime), cleaved-PARP1 (ab32064, Abcam), Bcl-2 (ab32124, Abcam), p21 (sc-6246, Santa Cruz), p53 (sc-126, Santa Cruz), HSP90 (ab282108, Abcam), GAPDH (60004-1-lg, Proteintech) and Vinculin (ab129002, Abcam) were used. Secondary antibodies HRP-linked anti-rabbit IgG antibody (7074S, Cell Signaling) and HRP-linked anti-mouse IgG antibody (7076S, Cell Signaling) were further used. Finally, proteins were visualized with the ECL System from Tanon (4600SF, China).
4
RIPA Lysis and Western Blot Analysis
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The radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) was used to extract total protein from OvCA cells. Determination of protein concentration was conducted with a BCA Protein Assay Kit (Thermo). 40 μg protein samples were loaded onto fresh sodium dodecyl sulfate polyacrylamide gel electrophoresis gel (10%). After isolating, these bands were then electro-transferred onto a PVDF membrane (Millipore, Massachusetts, USA) and then blocked with 5% skimmed milk, followed by incubation with the following primary antibodies against EIF5A2 (ab126733, 1:5000, Abcam, Cambridge, MA, USA), proliferating cell nuclear antigen (PCNA) (AF1363, 1:1000, Beyotime), Ki67(AF1738, 1:1000, Beyotime), cyclin D1 (ab16663, 1:200, Abcam), cyclin-dependent kinases 4 (CDK4) (ab108357, 1:1000, Abcam), cleaved caspase 3 (c-caspase 3) (ab32042, 1:500, Abcam), Bax (AF1270, 1:1000, Beyotime), Bcl-2 (AF1225, 1:1000, Beyotime), (E-cadherin (ab40772, 1:10,000, Abcam), N-cadherin (ab76011, 1:1000, Abcam) and Vimentin (AF1975, 1:1000, Beyotime), or anti-GAPDH (ab181602, 1:10,000, Beyotime) at 4 °C overnight and then incubated with secondary antibodies for 1 h. The integrated optical density was quantified with Image J software (NIH, Bethesda, MD, USA) after detecting the protein bands using the ECL system (Thermo).
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