Western blot experiment was manipulated following the previous study [30 (link)]. Briefly, the total protein of tissues from different fetuses was extracted according to the direction of RIPA lysis buffer (Beyotime, Beijing, China). The concentration of protein was measured by a BCA protein essay kit (Thermo Fisher Scientific, Waltham, USA) and then adjusted to 30 μg each hole for SDS-PAGE running. In this process, the proteins were subjected to immunoblotting analysis with the following antibodies: anti-EZH2 (1 : 1000, 5246S, Cell Signaling Technology, Danvers, Massachusetts, USA), anti-beta-tubulin (1 : 2000, GB11017, Servicebio, Wuhan, China), and HRP-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) (1 : 5000, SA00001-2, Proteintech, Rosemont, IL, USA). Finally, the membranes were developed with the ECL system (WBULS0500, Millipore Corporation, Billerica, USA).
Gb11017
Manufactured by Wuhan Servicebio Technology
Sourced in China
GB11017 is a laboratory equipment designed for cell culture applications. It provides a controlled environment for the growth and maintenance of cells. The product specifications and technical details are available upon request.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein essay kit, by Thermo Fisher Scientific (1 mentions)
Wbuls0500, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Sa00001 2, by Proteintech (1 mentions)
Skimmed milk, by BD (1 mentions)
Sds page gel, by Ipsen (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by CWBIO (1 mentions)
Hrp conjugated goat anti rabbit igg, by BBI Lifesciences (1 mentions)
0.45 μm polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Gb11001, by Wuhan Servicebio Technology (1 mentions)
Ab254360, by Abcam (1 mentions)
Ab255275, by Abcam (1 mentions)
Af7742, by Beyotime (1 mentions)
Gb114122, by Wuhan Servicebio Technology (1 mentions)
Gb113375, by Wuhan Servicebio Technology (1 mentions)
Ab184247, by Abcam (1 mentions)
Ab23707, by Abcam (1 mentions)
Ab214185, by Abcam (1 mentions)
Ab179812, by Abcam (1 mentions)
6 protocols using gb11017
1
Quantitative Western Blot Analysis of Fetal Tissue Proteins
2
Western Blot Analysis of PFKM Protein
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RIPA Lysis Buffer (CWBIO, Beijing, China) containing protease inhibitors was used to extract proteins. Samples were denatured by heating, proteins were separated via SDS–PAGE Gel (EpiZyme, Shanghai, China) electrophoresis and transferred onto PVDF membranes (Merck Millipore, Darmstadt, Germany), sealed with 5% skimmed milk (BD, Franklin Lakes, NJ, USA) for 2 h in a shaker at room temperature, washed with TBST, and incubated overnight at 4 °C with PFKM (Abcam, ab154804, 1:1000, Cambridge, UK) and Tubulin (Servicebio, GB11017, 1:1000, Wuhan, China) antibodies. Next, the samples were washed thrice with TBST and incubated with the secondary antibody (HRP-conjugated Goat Anti-Rabbit IgG, BBI Life Sciences D110058, 1:10,000, Shanghai, China) at 37 °C for 1 h. The target protein bands were visualized using chemiluminescent solution (CWBIO, Beijing, China), and the relative protein levels were quantified via Image J software.
3
Protein Separation and Quantification
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The human and mouse bone protein lysates were loaded into 10% SDS-PAGE gels, and the gels were cut into two parts. They were transferred into a 0.45-μm polyvinylidene difluoride membrane (Millipore) and separated. The large molecule protein CSPG4 (A3592, ABclonal) was processed at 300 mA for 3 h at 4°C with 10% methanol, and ITGA2B (A5680, ABclonal), tubulin (GB11017, Servicebio), and β-actin (GB11001, Servicebio) were processed at 300 mA for 1.5 h at 4°C with 20% methanol. The intensity of the protein was analyzed with ImageJ software.
4
Western Blot Analysis of Molecular Markers
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Western blotting was performed as previously described [26 (link)]. Proteins from the brain samples and cultured HT22 cells were lysed with RIPA lysis buffer. Equal amounts of protein (50 μg) were loaded onto a sodium dodecyl sulfate–polyacrylamide gel. The proteins were electrophoresed until they were fully separated and then transferred to a polyvinylidene difluoride membrane. The membrane was then blocked with 5% bovine serum albumin for 1 h at room temperature. The membranes were incubated with the following primary antibodies overnight at 4 ℃: anti-CCR2 (#12199, 1:1000, CST), anti-p-PI3K (#4228, 1:1000, CST), anti-PI3K (#AF7742, 1:1000, Beyotime), anti-AKT (#4691, 1:1000, CST), anti-pAKT (#4060, 1:1000, CST), anti-IL-1β (#ab254360, 1:1000, Abcam), anti-TNF-α (#ab255275, 1:1000, Abcam), anti-BAX (#GB114122, 1:1000, ServiceBio), anti-Bcl-2 (#GB113375, 1:1000, ServiceBio), anti-cleaved caspase 3 (#ab2302, CC3, 1:1000, Abcam), and anti-β-tubulin (#GB11017, 1:1000, ServiceBio). Suitable secondary antibodies (1:5000, Santa Cruz Biotechnology, Dallas, TX, USA) were selected and incubated with the membrane at room temperature for 1 h. The bands were then observed using an enhanced chemiluminescence reagent. Image J software (NIH, Bethesda, MD, USA) was used for density measurements and quantification.
5
Western Blot Analysis of Renal Protein Targets
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Renal tissues and cells were lysed to obtain the total proteins, quantifying with a BCA kit for concentration [17 (link)]. Then, protein samples were separated by SDS-polyacrylamide gels with suitable concentration and then transferred to PVDF membranes, and the membranes were blocked with 5% bull serum albumin and immunoblotted with primary antibodies overnight and subsequently incubated by a horseradish peroxidase-conjugated antibody. Primary antibodies NLRP3 (ab214185, Abcam), Keap1 (60027-1-Ig, Proteintech), Nrf2 (16396-1-AP, Proteintech), Drp1 (ab184247, Abcam), mfn2 (12186-1-AP, Proteintech), PINK1 (ab23707, Abcam) Parkin (ab179812, Abcam), p62 (ab109012, Abcam), β-actin (20536-1-AP, Proteintech), β-tubulin (GB11017, Servicebio, Wuhan), GAPDH (10494-1-AP, Proteintech), HMGB1 (DF7008,Affinity Biosciences), HO-1 (27282-1-AP, Proteintech), cleaved caspase 3 (9664S, Cell Signal Technology), and LC3-II (3868S, Cell Signal Technology) were used following the manufacturer's recommended dilutions. Image J software was used for semiquantitative calculation.
6
HMGB1 and RAGE Protein Levels
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The protein levels of HMGB1 and RAGE in the FL and sham cortices were assessed by Western blotting.
β-Tubulin was used as a loading control. Samples were selected from the operated side of the cortex, and total protein was extracted from three animals on P5, P15 and P30. The tissues were homogenized in lysis buffer and centrifuged at 4°C. Then, the supernatants were resolved via 12% SDS-PAGE, electrotransferred to a 0.45 µm PVDF membrane, and blocked for 2 h at room temperature with 5% nonfat dry milk in TBST. After overnight incubation with primary antibodies anti-HMGB1 mAb (GeneTex, GTX101277, 1:3000), anti-β-tubulin (Servicebio, GB11017, 1;3000) and anti-RAGE (Servicebio, GB11278, 1:1000)), the PVDF membrane was washed in TBST 3 times and incubated for 1 h with goat anti-rat IgG HRP (ANR02-1, 1:10000). Proteins were visualized by chemiluminescence with an ECL Western Blotting Substrate Kit (Merck-Millipore, WBKLS0100) according to the manufacturer's instructions. Densitometric quanti cation was performed with ImageJ software.
β-Tubulin was used as a loading control. Samples were selected from the operated side of the cortex, and total protein was extracted from three animals on P5, P15 and P30. The tissues were homogenized in lysis buffer and centrifuged at 4°C. Then, the supernatants were resolved via 12% SDS-PAGE, electrotransferred to a 0.45 µm PVDF membrane, and blocked for 2 h at room temperature with 5% nonfat dry milk in TBST. After overnight incubation with primary antibodies anti-HMGB1 mAb (GeneTex, GTX101277, 1:3000), anti-β-tubulin (Servicebio, GB11017, 1;3000) and anti-RAGE (Servicebio, GB11278, 1:1000)), the PVDF membrane was washed in TBST 3 times and incubated for 1 h with goat anti-rat IgG HRP (ANR02-1, 1:10000). Proteins were visualized by chemiluminescence with an ECL Western Blotting Substrate Kit (Merck-Millipore, WBKLS0100) according to the manufacturer's instructions. Densitometric quanti cation was performed with ImageJ software.
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