Proteins were extracted using a commercial kit (No.C510003, Sangon Biotech, China) according to the manufacturer's instructions. Primary antibodies rabbit anti-RUNX2 (ab23981, Abcam), rabbit anti-OSX (ab94744, Abcam), rabbit anti-ACAN (ab36861, Abcam), rabbit anti-OCN (23418-1-AP, ProteinTech, Wuhan, China), rabbit anti-ALP (ab83259, Abcam) and rabbit anti-PBX1 (ab97994, Abcam) were used (all at 1:1,000 dilution). The protein samples were separated on 10% SDS-PAGE gels and subsequently transferred to nitrocellulose filter membranes (Pall Corp.,Washington, NY) using the wet transfer blotting system (BioRad, Hercules, CA). After incubation, secondary antibody goat anti-rabbit-HRP (Pierce, USA) was used at 1:2,000 dilution. The proteins were then detected by chemiluminescence detection system (Millipore, USA). Anti-GAPDH was used as an endogenous control.
Wet transfer blotting system
Manufactured by Bio-Rad
Sourced in United States
The Wet transfer blotting system is a laboratory equipment designed for the transfer of proteins, nucleic acids, or other biomolecules from a gel to a membrane or other support matrix. The core function of this system is to facilitate the efficient and reliable transfer of these biomolecules, enabling further analysis and detection.
Automatically generated - may contain errors
Lab products found in correlation
Nitrocellulose filter membrane, by Pall Corporation (4 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ab83259, by Abcam (2 mentions)
Ab94744, by Abcam (2 mentions)
Ab23981, by Abcam (2 mentions)
Chemiluminescence detection system, by Merck Group (2 mentions)
No c510003, by Sangon (2 mentions)
Anti gapdh, by Abcam (2 mentions)
Ab36861, by Abcam (1 mentions)
Ab97994, by Abcam (1 mentions)
Anti ccr2, by Santa Cruz Biotechnology (1 mentions)
Anti p p65, by Abcam (1 mentions)
Ab191406, by Abcam (1 mentions)
Ab109526, by Abcam (1 mentions)
Ab19027, by Abcam (1 mentions)
Anti hif 1α, by Santa Cruz Biotechnology (1 mentions)
Anti nf κb, by Abcam (1 mentions)
Goat anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Protease inhibitor, by Wuhan Servicebio Technology (1 mentions)
Anti n cadherin, by ABclonal (1 mentions)
8 protocols using wet transfer blotting system
1
Protein Expression Analysis in Bone Cells
2
Western Blot Analysis of CCR1, CCR2, and p-p65
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The Western blot analysis was performed according to previously established methods [26 (link)]. Briefly, cells were collected and lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). All samples were normalized according to the protein concentrations and separated in 10% SDS-PAGE gels and then transferred to nitrocellulose filter membranes (Pall Corp., Washington, NY) using the wet transfer blotting system (Bio-Rad, Hercules, CA). The following antibodies were used for Western blotting: anti-CCR1, anti-CCR2 (Santa Cruz Biotechnology), anti-p-p65 (Abcam), and anti-GAPDH (Abcam) as an endogenous control. Goat anti-mouse CCR2 antibody was obtained from Santa Cruz Biotechnology. The gray levels of blots were analyzed using Image J software.
3
Protein Extraction and Western Blot Analysis of Bone Cell Markers
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Proteins were extracted using a commercial kit (No. C510003, Sangon Biotech, China) according to the manufacturer’s instructions. The following primary antibodies were used (all at a 1:1,000 dilution): rabbit anti-RUNX2 (ab23981, Abcam), rabbit anti-OSX (ab94744, Abcam), rabbit anti-OCN (23418-1-AP, ProteinTech, Wuhan, China), rabbit anti-ALP (ab83259, Abcam), rabbit anti-ACP5 (ab191406, Abcam), rabbit anti-ITBG3BP (ab192324, Abcam), rabbit anti-MMP9 (10375-2-AP, ProteinTech, Wuhan, China), rabbit anti-CTSK (ab19027, Abcam), rabbit anti-GAPDH (10494-1-AP, ProteinTech, Wuhan, China), rabbit anti-FOXO3 (10849-1-AP, ProteinTech, Wuhan, China) and rabbit anti-TAK1 (ab109526, Abcam). The protein samples were separated by 10% SDS–PAGE and subsequently transferred to nitrocellulose filter membranes (Pall Corp., Washington, NY) using a wet transfer blotting system (Bio-Rad, Hercules, CA). After incubation, a goat anti-rabbit-HRP secondary antibody (Pierce, USA) was applied at a 1:2,000 dilution. The proteins were then detected by a chemiluminescence detection system (Millipore, USA). Anti-GAPDH was used as an endogenous control. The uncropped scans of all blots were provided in the Source Data file.
4
Measuring HIF-1α and NF-κB in BMMSCs
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The total protein expression of HIF-1α and the total and nuclear level of protein expression of NF-κB in BMMSCs were measured by Western blotting. The Western blot analysis was performed according to previously established methods [22 (link)]. Briefly, BMMSCs were collected and lysed in M-PER Mammalian Protein Extraction Reagent (Pierce, Rockford, IL). All samples were normalized according to the protein concentrations and separated in 10% SDS-PAGE gels and then transferred to nitrocellulose filter membranes (Pall Corporation, Washington, NY) using the wet transfer blotting system (Bio-Rad, Hercules, CA). The following antibodies were used for Western blotting: anti-HIF-1α (Santa Cruz Biotechnology), anti-NF-κB (Abcam), and anti-GAPDH (Abcam). Goat anti-rabbit secondary antibody was obtained from Santa Cruz Biotechnology. The gray levels of blots were analyzed using ImageJ software.
5
Western Blot Analysis of Cell Lines
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HT29 and CT26.WT were cultured in a six-well plate for 48 h; total protein was extracted using RIPA buffer (Servicebio, G2002, China) containing protease inhibitors (Servicebio, G2006, China) and quantified using BCA kit (Thermo Fisher Scientific, USA). For each sample, 25 μg total protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane using a wet transfer blotting system (BioRad, Hercules, CA), antibodies such as anti-IRF-1 (Cell Signaling Technology, #8474, USA), anti-IRF-2 (Abcam, ab1274744, USA), anti-E-cadherin (ABclonal, A3044, China), anti-N-cadherin (ABclonal, A0433, China), anti-Vim (ABclonal, A11423, China) were used for western blotting; anti-histone (Abcam, USA) was used as an endogenous control.
6
Northern Blot Analysis of tRNA Expression
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Total RNA (1.5 μg) isolated from WT and MTU1 KO cells was separated with 7 M urea/TBE/15% PAGE at 150 V. The gel was then stained with SYBR Gold (Invitrogen) to assess the RNA quality and transferred to a nylon membrane (Millipore) by a wet transfer blotting system (Bio-Rad) on ice at 50 V for 80 min. The membrane was then crosslinked with UV light at 1200 × 100 mJ/cm2 (HL-2000 Hybrilinker, UVP), soaked in prehybridization buffer (6× SSC, 0.1% SDS and 1× Denhardt's Solution) and incubated in a 42°C water bath for 1 h. After incubation, the membrane was hybridized with DIG (Roche)-labeled tRNA probes in hybridization buffer (900 mM NaCl, 90 mM Tris–HCl pH 8, 6 mM EDTA and 0.3% SDS) and incubated overnight at 50°C. The membrane was then washed with 1× SSC, blocked using a DIG wash and block buffer set (Roche), and detected with anti-DIG alkaline phosphatase Fab fragments (Roche) and CDP-Star (Roche). The membranes were imaged by ImageQuant (GE Healthcare). Probes used for northern blotting are listed in Supplementary Table S2 .
7
Immunoblotting Protein Detection Protocol
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For immunoblotting, proteins were transferred to PVDF membranes, which were previously activated with 96% ethanol for 2 min, overnight 12–20 V at room temperature using the wet blotting transfer system of Bio-Rad (Hercules, CA, USA). For the immunological detection of the proteins of interest, the following incubation steps were carried out:
1 h incubation at room temperature with blocking buffer [5% w/v non-fat dry milk in 1× TBS (Tris buffered saline, 0.05 M Tris and 0.15 M sodium chloride, pH 7.6), 0.05% Tween®] in agitation.
Overnight incubation at 4 °C on rocking platform with the primary antibody of interest (
3 washes of 10 min with 1× TBS containing 0.05% Tween®.
1 h incubation at room temperature on rocking platform with the corresponding secondary antibody (
3 washes of 10 min with 1× TBS containing 0.05% Tween®.
8
Western Blot Analysis of pSMAD1/5/8
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Snap-frozen renal cortex was homogenized and lysed using NP-40 lysis buffer containing Na-orthovanadate, Na-fluoride, and complete protease inhibitor cocktail. Lysates were spun down, and pellets were discarded. Total protein concentration in the supernatant was measured using BCA (Pierce Thermo, Rockford, IL). Twenty micrograms of protein was boiled with Laemmli/DTT and run for 90 min on 10% SDS-PAGE gels (Bio-Rad). Gels were subsequently blotted for 90 min on polyvinylidene difluoride membrane using a wet blotting transfer system (Bio-Rad). For pSMAD1/5/8 analysis, membranes were incubated with primary antibody (pSMAD1/5/8, 1:2,000, Cell Signaling Technology; SMAD1/5/8, 1:1,000, Santa Cruz Biotechnology) in TBS-Tween containing 3% BSA overnight. After a thorough rinsing, membranes were incubated with secondary horseradish peroxidaseconjugated antibody and imaged using chemiluminescence substrate (GE Healthcare Life Sciences, Buckinghamshire, UK).
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