Protein extraction and immunoblotting were performed as described in our previous study [28 (link)]. Hearts and kidneys from GHRKO piglets and wild-type piglets were used to evaluate GHR protein levels using western blot analysis. In brief, tissues were lysed using RIPA lysis buffer (Bestbio, China) with protease inhibitors at 4 °C. After lysis, supernatants were collected by centrifugation at 12,000× rpm for 15 min at 4 °C. Equal amounts of protein (60 μg) and a protein weight marker were separated by SDS-PAGE. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes and incubated with primary antibodies against GHR (1:1000 v/v, ab202964, Abcam, Cambridge, England) and β-actin (1:5000 v/v, Sigma-Aldrich;) at 4 °C overnight. After incubation, the membranes were washed and reacted with anti-mouse or anti-rabbit secondary antibodies (R&D, USA). The membranes were then incubated with enhanced chemiluminescence (ECL) (Easysee Western Blot Kit, China) and visualized with an imaging system (Bio-Rad).
Easysee western blot kit
Manufactured by Bio-Rad
Sourced in China
The Easysee Western Blot Kit is a laboratory equipment product designed for the detection and analysis of proteins using the Western blot technique. The kit provides the necessary components to perform this analytical procedure, including reagents, membranes, and detection methods. The core function of the Easysee Western Blot Kit is to facilitate the visualization and quantification of specific proteins within a sample.
Automatically generated - may contain errors
Lab products found in correlation
Imaging system, by Bio-Rad (2 mentions)
Anti mouse or anti rabbit secondary antibodies, by R&D Systems (2 mentions)
Ripa lysis buffer, by BestBio (2 mentions)
Universal hood 2, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ab202964, by Abcam (1 mentions)
β actin, by Merck Group (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Phospho pi3k, by Cell Signaling Technology (1 mentions)
Phospho akt, by Proteintech (1 mentions)
Gapdh, by Merck Group (1 mentions)
5 protocols using easysee western blot kit
1
Western Blot Analysis of GHR Protein
2
GGTA1 Protein Analysis in Piglet Pancreas
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Protein extraction and immunoblotting were performed as previously described in our previous study [25 (link)]. The pancreas tissue from GTKO piglets and cloned piglet derived from unmodified original donor cells were used to evaluate GGTA1 protein levels using western blotting. In brief, pancreas tissues were lysed in RIPA lysis buffer (Bestbio, China) with protease inhibitors at 4 °C. After lysis, the supernatants were obtained by centrifugation at 13,800 × g for 15 min at 4 °C. Equal amounts of protein (70 μg) were run on SDS-PAGE gel, along with molecular weight marker. After electrophoresis, the proteins were transferred to PVDF membranes and reacted with primary antibodies against GGTA1 (ALX-801-090-1, Enzo, Lausen, Switzerland; 1:15) and β-actin (anti-β-actin, Sigma-Aldrich; 1:2000) at 4 °C overnight. After incubation, the membranes were washed and incubated with anti-mouse secondary antibodies (R&D, USA). The membranes were incubated with the ECL (Easysee Western Blot Kit, China) and visualized with an Imaging System (Bio-Rad, Universal Hood II, USA).
3
Muscle MSTN Protein Detection by Western Blot
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We selected the muscle tissues described above, detected the protein of MSTN by western blotting compared with WT lambs. Muscle tissues were lysed in RIPA lysis buffer (Beyotime, China) with protease inhibitors at 4 °C. After lysis, supernatants were obtained by centrifugation at 14,000 × g for 15 min at 4 °C. The protein (50 μg) were separated using SDS-PAGE. After electrophoresis, the proteins were transferred to PVDF membrane and reacted with primary antibodies against MSTN (anti-MSTN, 1:1000, Thermo Scientific) and β-actin (anti-β-actin, 1:5000, Sigma-Alderich) at 4 °C overnight. After incubation, membranes were washed and incubated with anti-mouse or anti-rabbit secondary antibodies (R&D, USA). The membranes were developed using the ECL detection system (Easysee Western Blot Kit, China) and visualized with an Imagining System (Bio-Rad, Universal Hood II, USA).
4
Western Blot Analysis of Cellular Proteins
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For protein samples, we used RIPA (Solaribio, China) to extract total protein from cells and tissue samples mentioned above on the ice. Then equal amount of proteins in each sample was separated by 10–12% SDS-PAGE (Servicebio, China) and transferred onto polyvinylidene fluoride membranes (Millipore, United States). The membrane was treated with a blocking buffer, which consisted of TBST mixed with 5% skim milk, and this process was conducted at room temperature for 1 h. We used TIGD1 (1:1000, 13833-1-AP, proteintech, China), AKT (1:1000, 10176-2-AP, proteintech, China), Phospho-AKT (1:1000, 28731-1-AP, proteintech, China), PI3K (1:1000, 20584-1-AP, proteintech, China), Phospho-PI3K (1:1000, 17366, Cell Signaling Technology, United States), GAPDH (1:100000, 60004-1-Ig, proteintech, China), Beta Actin (1:20000, 66009-1-Ig, proteintech, China) antibodies for incubation at 4°C overnight. Following three washes with TBST, the membrane was subjected to incubation with secondary antibodies. Subsequently, it was treated with enhanced chemiluminescence (ECL) (Easysee Western Blot Kit, China), and digital blot images were captured using an imaging system (Bio-Rad, United States).
5
Western Blot Analysis of P53 and P21
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FFCs were cultured at a density of 1 × 106/well in a 10-cm plate and treated with or without DOX (100 μmol/L) for 24 h. The cells were harvested and lysed in RIPA lysis buffer (Beyotime, China) with protease inhibitors at 4 °C. After lysis, supernatants were obtained by centrifugation at 14,000×g for 15 min at 4 °C. The proteins (50 μg) were separated using SDS-PAGE. After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes and reacted with primary antibodies against P53 (Imaxgen), P21 (Eptomics) and GAPDH (Sigma). After incubation, membranes were washed and incubated with anti-rabbit secondary antibodies (R&D, USA). The membranes were developed using the ECL detection system (Easysee Western Blot Kit, China) and visualized with an imagining system (Bio-Rad, Universal Hood II, USA).
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