Heart tissue and primary rat cardiomyocytes were lysed in RIPA lysis buffer with 1% protease inhibitor (Beyotime Institute of Biotechnology). A BCA kit was used to determine the protein concentration in the supernatant (Beyotime Institute of Biotechnology). In total, ~50 µg heart tissue lysate or 20 µg cell lysate was used for 12% SDS-PAGE, and proteins were then transferred to an FL membrane (MilliporeSigma) at 4˚C for 1.5 h. After blocking with 5% non-fat milk powder (Beyotime Institute of Biotechnology) at room temperature for 1.5 h, the following primary antibodies were added and incubated overnight at 4˚C: SOD2 (1:1,000), pro/cleaved caspase-3 (1:1,000), Bax (1:1,000), NOX2 (1:2,000) and Bcl-2 (1:1,000) were. Subsequently, HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibodies (1:10,000; Thermo Fisher Scientific, Inc.; cat. no. 31460) were added and incubated at room temperature for 1.5 h. The western blotting results were analyzed using BeyoECL Plus (Beyotime Institute of Biotechnology) and Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.). The specific protein expression levels were normalized to that of GAPDH.
Non fat milk powder
Manufactured by Beyotime
Sourced in China
Non-fat milk powder is a dehydrated milk product. It is produced by removing the fat content from milk and then drying the remaining solids into a powdered form. The core function of non-fat milk powder is to provide a concentrated source of milk proteins, carbohydrates, and minerals.
Automatically generated - may contain errors
Lab products found in correlation
Hrp conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (2 mentions)
Fl membrane, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Beyoecl plus, by Beyotime (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Bca kit, by Beyotime (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Ab40772, by Abcam (1 mentions)
Ab76011, by Abcam (1 mentions)
Ab302958, by Abcam (1 mentions)
Ab278545, by Abcam (1 mentions)
Ab238477, by Abcam (1 mentions)
Ab206414, by Abcam (1 mentions)
Ab32042, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
Gvwp02500, by Merck Group (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Ab92547, by Abcam (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
5 protocols using non fat milk powder
1
Cardiac Protein Quantification and Analysis
2
Protein Analysis via Western Blot
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Protein samples were isolated using RIPA buffer (Sigma, R0278, USA) with PMSF, and concentrations determined via BCA assay (Nanjing Jiancheng Bioengineering Institute, #A045-4-1, China). SDS-PAGE (10%) and subsequent wet transfer onto membranes (Milipore, GVWP02500, USA) preceded antibody incubations. Blocking utilized TBST with non-fat milk powder (Beyotime, #P0222, China), and detection was via enhanced chemiluminescence (Bio-Rad, #32106, USA). Antibodies against GDF15, β-actin, PI3K, AKT, cleaved caspase-3, Bax, N-cadherin, E-cadherin, vimentin, and GAPDH (all from abcam) were applied. GDF15 (1:1000, ab206414, abcam), β-actin (1:3000, ab5694, abcam), p-PI3K (1:2000, ab278545, abcam), PI3K (1:2000, ab302958, abcam), AKT (1:2000, ab238477, abcam), p-AKT (1:2000, ab81283, abcam), cleaved caspase-3 (1:1000, ab32042, abcam), bax (1:1000, ab32503, abcam), N-cadherin (1:2000, ab76011, abcam), E-cadherin (1:2000, ab40772, abcam), vimentin (1:1000, ab92547, abcam), GAPDH (1:3000, ab9485, abcam).
3
Cardiac mitochondrial protein analysis
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The mitochondria and cytoplasm of rat ventricular tissue and primary rat cardiomyocytes were separated according to the instructions of tissue or cell mitochondrial extraction kit (Beyotime Institute of Biotechnology). A BCA kit was used to determine the protein concentration thereafter. Then, ~30 µg heart tissue or cell lysate was loaded per lane onto 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, after which proteins were transferred to an FL membrane (MilliporeSigma) at 4˚C for 2 h. After blocking with 5% blocking protein powder (non-fat milk powder; Beyotime Institute of Biotechnology) at room temperature for 2 h, the following primary antibodies were added and incubated overnight at 4˚C: Cyt C (1:1,000), pro/cleaved caspase-3 (cat. no. 14220S/cat. no. 9664S; 1:1,000), cleaved caspase-9 (cat. no. 9507; 1:1,000), COX IV (cat. no. 4850; 1:1,000) and GAPDH (cat. no. 10494-1-AP; 1:5,000). Subsequently, HRP-conjugated goat anti-rabbit IgG (H+L) secondary antibodies (1:10,000; Thermo Fisher Scientific, Inc.; cat. no. #31460) were added and incubated at room temperature for 1.5 h. The results of western blotting were analyzed using BeyoECL Plus (Beyotime Institute of Biotechnology) and Image Lab software (version 5.2.1; Bio-Rad Laboratories, Inc.). The specific protein expression levels were normalized to GAPDH or COX IV.
4
Western Blot Analysis of Key Proteins
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The specific steps of Western blot were performed as described previously [33 (link)]. First, the radioimmunoprecipitation assay buffer (RIPA) was used to obtain proteins, followed by protein concentration assay using bicinchoninic acid (BCA) assay kit (Beyotime, China). Next, proteins were then separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The separated proteins were transferred to polyvinylidene (PVDF, Bio-Rad, USA) membrane. Then, 5% nonfat milk powder (Beyotime, China) was used to enclose the membrane at room temperature for 1 h. After blocking, the primary antibodies were introduced to incubate overnight at 4°C. After incubation, TBST (Beyotime, China) was used to wash the membrane, which was then incubated at room temperature with secondary antibody for 4 h. Finally, ECL hypersensitive chemiluminescence kit was used for protein color development, and pictures were taken with the visualizer. The primary antibodies were sourced from Abcam (UK): rabbit anti-human WTAP (ab195380), FLNA (ab76289), LC3I/II (LC3B, ab51520), β-actin (ab8227), p62 (ab109012). The secondary antibody was goat anti-rabbit IgG (ab6721), which was purchased from Abcam (UK).
5
Unraveling TNBC Proteome: Native PAGE Analysis
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Total protein was isolated from TNBC samples (tumour tissues, paired normal adjacent tissues and cell lines) using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was separated by 10% SDS‐PAGE (25 μg protein per lane), and the protein was transferred with PVDF membrane (MilliporeSigma). Blocking of PVDF membrane was performed with 5% nonfat milk powder (Beyotime). To detect the expression of PKM2 protein in different conformations, the total protein extracted from the sample was not thermal denatured and was denatured using a non‐denatured gel sample loading buffer (Beyotime). Next, protein electrophoresis was performed using native PAGE running buffer (Beyotime). The PVDF membranes were probed at 4°C overnight with antibodies against METTL14 (1:1000; Abcam), PKM2 (ProteinTech Group, Inc.), DGCR8 (1:1000; Abcam), TRIM9 (1:1000; Abcam) and β‐actin (1:5000; Abcam). Finally, protein expression was analysed by chemiluminescence reagents (Hyperfilm ECL).
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