PMSF and phosphatase inhibitor (Topscience, China) was added to the RIPA buffer (SparkJade Biotechnology, China) for protein extraction from THP-1 cells and mouse tissues. And then the concentration of each sample was determined using the BCA assay kit (Beyotime, China). The protein separation was performed using Tris-glycine gels (Solarbio, China), followed by transfer onto polyvinylidene difluoride membranes (Millipore, USA). Following sealing with TBST containing 5% skim milk for 1 hour at room temperature, the membranes were subjected to overnight incubation at 4°C with primary antibodies (diluted 1:1000). Subsequently, they were further incubated with HRP-coupled secondary antibodies (diluted 1:3000) for 1 hour. Finally, blots were imaged in the imaging system (US Azure, C300) by using ECL Super Kit.
Ripa buffer
Manufactured by Sparkjade
Sourced in China
RIPA buffer is a commonly used lysis buffer for the extraction and solubilization of proteins from cells and tissues. It contains a mixture of ionic and non-ionic detergents that help to disrupt cell membranes and release intracellular proteins. The buffer also includes other components that help to maintain protein stability and prevent degradation.
Automatically generated - may contain errors
Lab products found in correlation
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Bca assay kit, by Beyotime (1 mentions)
Gapdh, by Transgene (1 mentions)
Vimentin, by Proteintech (1 mentions)
E cadherin n cadherin, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Yeasen (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by neoFroxx (1 mentions)
Pvdf membrane, by Sparkjade (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Wuhan Servicebio Technology (1 mentions)
Ab32445, by Abcam (1 mentions)
Ab188161, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Bioss Antibodies (1 mentions)
Ac002, by Abcam (1 mentions)
P erk, by ZenBio (1 mentions)
4 protocols using ripa buffer
1
Protein Analysis in THP-1 Cells and Mouse Tissues
2
Western Blot Analysis of Tissue Proteins
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Tissue proteins were extracted using the RIPA Buffer (SparkJade) and protease inhibitor (SparkJade). Proteins were denatured by boiling in SDS-PAGE protein loading buffer (SparkJade). Total proteins were subjected to 5–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to immobilon-P PVDF membranes (MILLIPORE) using an immunoblot transfer buffer. After incubation at 5% BSA (Biofroxx) for 1 h at room temperature, membranes were incubated with primary antibodies overnight at 4°C. Antibodies purchased from ZEN-BIOSCIENCE (E-cadherin, N-cadherin), Cell Signaling (Vimentin), Proteintech (MMP13), Transgen (GAPDH) were used according to manufacturer’s recommendations. The next day, membranes were incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies (SparkJade or ZEN-BIOSCIENCE) for 1 h at room temperature. After three washing steps, blots were stained using a chemiluminescence system (ECL, YEASEN) and exposed to X-ray film. Details of antibodies are shown in Supplementary Table 1 .
3
Western Blot Analysis of Angiogenic Factors
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Endometrial tissues were lysed using RIPA buffer (Sparkjade, Jinan, China) and protein concentration evaluated by a BCA assay kit (Thermo Fisher Scientific). Equal concentrations of proteins were then separated on 10% SDS-PAGE gels, transferred onto polyvinylidene difluoride fluoride (PVDF) membrane (Sparkjade, Jinan, China) that were then blocked for 1 h using 5% skimmed milk in Tris-buffered saline with 0.05% at room temperature (RT). Thereafter, overnight incubation of membranes was done with rabbit primary antibodies against HIF1α, VEGFA, and VEGFR2 (all from Sangon Biotech, Shanghai, China) at 4°C. They were then incubated for 2 h with HRP-conjugated secondary antibodies at RT and signal developed by an ECL kit (Thermo Fisher Scientific). GAPDH was used as loading control. Image J (NIH, MD, USA) was used to measure band intensities.
4
Western Blot Analysis of Protein Expression
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The cells were lysed in RIPA buffer (Sparkjade, Jinan, China) supplemented with protease inhibitor cocktail (Servicebio, Wuhan, China) and phosphatase inhibitors A and B on ice for 30 min. The lysates were centrifuged for 15 min at 12,000 rpm, and their protein content was measured. Equal amounts of protein per sample were resolved on 10% SDS-PAGE gels and transferred to a PVDF membrane. After blocking with 5% BSA in TBST for 2 h at room temperature, the membranes were incubated overnight with antibodies targeting GAPDH (Abclonal, Wuhan, China, AC002), RRS1 (Abcam, Cambridge, UK, AB188161), AEG-1 (Abcam, Cambridge, UK, ab227981), ABCG2 (ZenBio, Research Triangle Park, NC, USA, R26465), MDR1 (Proteintech, Wuhan, China, 22336-1-AP), ERK (ZenBio, NC, USA, 340373), p-ERK (ZenBio, NC, USA, 340767), BAX (CST, Danvers, MA, USA, #89477), Bcl-2 (CST, Danvers, MA, USA, #3498), BAD (Abcam, Cambridge, UK, ab32445), p-BAD (Abcam, Cambridge, UK, ab129192) and ubiquitin (Proteintech, Wuhan, China, 10201-2-AP) at 4 °C. The membranes were washed thrice with TBST and then incubated with HRP-conjugated secondary antibody (1:1000; Bioss, Beijing, China, bs-0295G-HRP), followed by three more washes with TBST. The positive bands were visualized via ECL (MDBio, Taiwan, China).
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