Samples from each group were lysed in modified RIPA lysis buffer (Beyotime Biotech, China), which contained protease inhibitor cocktail (Sigma), and then placed on ice for 30 min. Then, samples were centrifuged at 13000 rpm and 4 °C for 30 min. Protein concentrations were determined using a BCA protein assay kit (Pierce, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal quantities of protein were loaded onto 12.5% SDS-PAGE for electrophoresis, followed by transfer to PVDF membranes (Millipore). After blocking with 5% nonfat milk for 2 h, membranes were immunoblotted with rabbit anti-human polyclonal antibodies against p53 (1:5000, Santa Cruz), p21 (1:1000, CST) and PUMA (1:1000, Abcam) overnight at 4 °C. Then, appropriate secondary antibodies (goat-anti-rabbit antibody, 1:10000; ZSGB-BIO, Beijing, China) were used for incubating the blots for 1 h at 37 °C. GAPDH was used as a control (1:5000; Zen-BioScience, Chengdu, China). A western blotting luminol reagent (Millipore, MA, USA) was used to visualize the immunoreactive bands.
Modified ripa lysis buffer
Manufactured by Beyotime
Sourced in China
Modified RIPA lysis buffer is a versatile reagent used for the extraction and solubilization of proteins from biological samples. It is a buffer solution that contains a combination of detergents, salts, and other components designed to efficiently lyse cells and release their protein contents.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Gapdh, by ZenBio (1 mentions)
Western blotting luminol reagent, by Merck Group (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Ecl detection reagent, by Merck Group (1 mentions)
Protease inhibitors cocktail, by Roche (1 mentions)
Skimmed milk, by Merck Group (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Beyotime (1 mentions)
Enhanced bca protein assay kit, by Beyotime (1 mentions)
Ab182733, by Abcam (1 mentions)
Ab32064, by Abcam (1 mentions)
Ab16051, by Abcam (1 mentions)
Ab110304, by Abcam (1 mentions)
Ab16663, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Ab32370, by Abcam (1 mentions)
Ab16048, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
5 protocols using modified ripa lysis buffer
1
Western Blot Analysis of Cell Signaling
2
Western Blot Analysis of SIRT1 Expression
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Total proteins from breast cancer cells were lysed in modified RIPA lysis buffer (Beyotime, China) with freshly added protease inhibitors cocktail (Roche Diagnostics, Basel, Switzerland) and quantified by a BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Then, 20 µg of total cellular extracts were separated by 10% SDS-PAGE and immobilized on polyvinylidene fluoride membrane (PVDF; EMD Millipore, Billerica, MA, USA). Following blocked by 5% skimmed milk (Sigma) for 2 h, the membrane was probed with primary antibodies against sirt1 and β-Actin (Abcam, Cambridge, MA, USA) overnight at 4 °C. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-conjugated goat-anti-mouse IgG (Santa Cruz biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature. The protein bands were visualized using ECL detection reagent (Millipore, Billerica, MA, USA).
3
Protein Expression Analysis in Cell Fractions
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Total cell protein was extracted with modified RIPA lysis buffer (Beyotime). The nuclear and cytosolic cell fractions were extracted with the Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime). The protein concentration was measured with an Enhanced BCA Protein Assay Kit (Beyotime). An equal amount of protein was used for sodium dodecyl sulphate polyacrylamide gel electrophoresis and polyvinylidene fluoride membrane transfer. Primary antibodies for cleaved poly (ADP-ribose) polymerase (PARP, ab32064), Bcl-2 (ab32124), B-cell lymphoma-extra large (Bcl-xL, ab32370), B-cell associated X protein (Bax, ab182733), β-catenin (ab16051), cyclin D1 (ab16663), SIRT1 (ab110304), glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab181206), Lamin B1 (ab16048) and β-actin (ab8226) (all purchased from Abcam) were incubated with membranes. The immunoreactive bands were analyzed with an enhanced chemiluminescent substrate (Pierce, Rockford, IL, USA).
4
Western Blot Analysis of Inflammatory Markers
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The protein extracts were obtained using modified RIPA Lysis Buffer (Beyotime, Haimen, China). Equal amount of protein samples was loaded onto SDS-PAGE and blotted onto PVDF membranes. After blocking in skimmed milk, the membranes were probed with primary antibodies against iNOS (ab205529, 1:1,000, Abcam, UK), TNF-α (ab255275, 1:1,000, Abcam), IL-10 (ab189392, 1:1,000, Abcam), Arg-1 (1:5,000, 16001-1-AP, Proteintech, Wuhan, China), ISG15 (1:1,000, 15981-1-AP, Proteintech), STAT1 (1:2,000, 10144-2-AP, Proteintech), p-STAT1 (1:1,000, ab109461, Abcam), p-JAK (bs-4163R, 1:500, Bioss, Beijing, China), JAK (bs-1439R, 1:500, Bioss), β-actin (1:1,000, 20536-1-AP, Proteintech) at 4°C overnight, followed by incubation with secondary antibody. Signals were detected using the BeyoECL Moon (Beyotime).
5
Western Blot Analysis of Transfected A549 Cells
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After 48 h transfected with siRNA, A549 cells were undergone for a Western blot assay. [40 (link), 41 (link)] Cells were collected and homogenized in modified RIPA lysis buffer (Beyotime, China) with 0.5% protease inhibitor cocktail (Roche Diagnostics) and stayed on ice for 10 min. After centrifuging at 12,000 rpm, the supernatants were obtained and sonicated for 15 sec. The supernatants were quantified by BCA assay (BioRad Laboratories, USA). Then, the extracts were boiled with 5X loading buffer [Chunfeng Lv, China]. Twenty 20 μg total protein were electrophoresed through 10% SDS-PAGE gels and, then transferred onto the PVDF membrane. After blocking nonspecific binding sites with 5% milk in PBS for 1 h, membranes were incubated with primary antibodies of GAPDH (cat no. 5174; 1:2,000; Cell Signaling Technology, Inc; USA); RRBP1 (cat no. ab224354, 1:1000, Abcam, UK) or SLC39A7 (cat no. ab254566; 1:1000; Abcam, UK) overnight at 4° C. Then, the membranes were cultured with secondary antibodies conjugated with horseradish peroxidase at room temperature for 1 h. Last, the membranes were treated with ECL reagent for developing films at darkness.
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