The ovaries were homogenized using 500 μL ice-cold RIPA supplemented with 1 mM phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China) and proteinase inhibitors (Beijing Solarbio Science & Technology Co., Beijing, China). Total protein concentrations were determined using a BCA protein assay kit (Nanjing Jiancheng Bioeng Ins, Nanjing, China). A total of 24 μg protein was separated with SDS-PAGE and transferred to polyvinylidene difluoride (PVDF ) membrane (0.22 μm, Millipore, Bedford, USA). After blocking with 5% skim milk, the PVDF membrane was incubated with corresponding primary antibodies including rabbit anti-VEGF (1:500), rabbit anti-p-p38, rabbit anti-Raf1, rabbit anti-PKG-I (1:500, Hangzhou HuaAn Biotechnology Co., Hangzhou, China), rabbit anti‐CCND1 (1:200), rabbit anti‐CDK2 (1:200, Boster Bioengineering Co., Wuhan, China), mouse anti-PCNA (1:1000), or mouse anti‐β‐actin (1:1000, Abcam, Cambridge, UK) at 4°C overnight. Then the PVDF membrane was incubated with the secondary antibody at room temperature for 1 h. Immunological signals were detected by an enhanced chemiluminescence kit (Bio-Rad, Hercules, USA) using a ChemiScope 3400 Mini machine (Clinx, Shanghai, China). The band intensities were quantified with Quantity One Software and the results were normalized to β-actin.
Chemiscope 3400 mini machine
Manufactured by Clinx
Sourced in China
The ChemiScope 3400 Mini is a compact and versatile laboratory instrument designed for various analytical applications. It features a high-resolution camera and advanced image processing capabilities to capture and analyze digital images. The ChemiScope 3400 Mini is a self-contained unit capable of performing tasks such as gel documentation, blot imaging, and basic fluorescence detection.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Proteinase inhibitor, by Solarbio (2 mentions)
Phenylmethanesulfonyl fluoride, by Beyotime (2 mentions)
Bca protein assay kit, by Nanjing Jiancheng (2 mentions)
Mouse anti pcna, by Abcam (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Mouse anti β actin, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Clarity ecl western blot substrate kit, by Bio-Rad (1 mentions)
Ab5714, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit or anti mouse igg sc 2004 or sc 2005, by Santa Cruz Biotechnology (1 mentions)
P00138, by Beyotime (1 mentions)
Sc 28266, by Santa Cruz Biotechnology (1 mentions)
St506, by Beyotime (1 mentions)
Biotinylated goat anti rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (1 mentions)
Ecl kit, by Bio-Rad (1 mentions)
Df7576, by Affinity Biosciences (1 mentions)
4 protocols using chemiscope 3400 mini machine
1
Western Blot Analysis of Ovarian Proteins
2
Protein Extraction and Western Blot Analysis
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Proteins were extracted using an ice-cold RIPA lysis buffer (P00138, Beyotime, Nanjing, China) with proteinase inhibitor (ST506, Beyotime, Nanjing, China). Equal amounts of proteins were measured using an BCA protein assay kit (A045-3, Jiancheng, Nanjing, China). Proteins were separated by electrophoresis and then electronically transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, USA) using a BioRad system (BioRad, Hercules, USA). The membrane was blocked in 5% skimmed milk at room temperature for 2 h and subsequently incubated overnight at 4°C with corresponding primary antibodies, rabbit anti-BCL2 (1:200), mouse anti-GRP78 (1:200), rabbit anti-LC3β (1:100, sc-28266, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-CYP11A1 (1:200, CSB-PA006389LA01HU, CusAb, Wuhan, China), mouse anti-BAX (1:100, ab5714) and mouse anti-β-actin (1:1000, ab8226, Abcam, Cambridge, UK). Horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (sc-2004 or sc-2005, Santa Cruz Biotechnology, Dallas, USA) were then used to detect proteins using Clarity ECL Western Blot Substrate kits (BioRad, Hercules, USA) and exposed using a ChemiScope 3400 Mini machine (Clinx, Shanghai, China). Protein quantification was performed using densitometry analyses on Quantity One Software.
3
Western Blot Analysis of CALB1 and ATP2A1
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The uterine tissues, duodenum, and kidney were homogenized in 500 μL of ice-cold radioimmunoprecipitation lysis buffer supplemented with 1 mmol/L phenylmethanesulfonyl fluoride (Beyotime, Shanghai, China) and proteinase inhibitors (Beijing Solarbio Science & Technology Co., Beijing, China). Bicinchonininc acid protein assay kit (Nanjing Jiancheng Bioeng Ins, Nanjing, China) was used to determine total protein concentration. A total of 24 μg of protein was separated with SDS-PAGE and transferred to polyvinylidene difluoride (PVDF ) membrane (0.22 μm, Millipore, Bedford). After blocking with 5% skim milk, the PVDF membrane was incubated with corresponding primary antibodies including rabbit anti-CALB1 (1:1,000, ET1702-54) and rabbit anti-ATP2A1 (1:500, ER1803-34, Hangzhou HuaAn Biotechnology Co., Hangzhou, China) at 4°C overnight. Then, the PVDF membrane was incubated with biotinylated goat anti-rabbit IgG (1:100, Santa Cruz Biotechnology, Dallas, TX) at room temperature for 1 h. Immunological signals were detected by an enhanced chemiluminescence kit (Bio-Rad, Hercules, CA) using a ChemiScope 3400 Mini machine (Clinx, Shanghai, China). The band intensities were quantified using Quantity One Software, and the results were normalized to β-actin.
4
Western Blot Analysis of ER Stress Markers
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The SWFs were fully lysed by an ice-cold RIPA solution, and the SWF-lysate was centrifuged at 12000 rpm for 20 min at 4°C. Protein concentration in the supernatants was measured by a BCA protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Samples of 20 μg of protein were applied to SDS-PAGE glue, and the protein was transferred to a methanol-activated polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA) after running for 30 min at 80 V and 90 min at 120 V. After blocking PVDF with 5% skim milk, the immunoassay was detected with the corresponding primary antibodies and then incubated with the secondary antibodies. The bands were visualized by an enhanced chemiluminescence (ECL) kit (Bio-Rad, Hercules, USA). A ChemiScope 3400 Mini machine (Clinx, Shanghai, China) was used to detect the signal intensity. The antibodies used for Western blot were as follows: mouse anti-GRP78 (1 : 1000, sc-376768), CHOP (1 : 1000, sc-46661, Santa Cruz Biotechnology, Santa Cruz, USA), rabbit anti-caspase12 (1 : 1000, NBP1-76801), PERK (1 : 1000, NBP1-80930, Novus, USA), phospho-PERK (1 : 1000, DF7576, Affinity Biosciences, OH, USA), anti-ATF4 (1 : 1000, ET1612-37), anti-caspase3 (1 : 100, ET1602-39), and mouse anti-calreticulin (1 : 100, EM1701-61, HuaBio, Hangzhou, China).
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