The tissues were homogenized and the total proteins were extracted in radioimmunoprecipitation lysis buffer (Beyotime Biotechnology, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride. The proteins were quantified using the BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Western blotting was performed as previously described.19 (link) Briefly, the proteins were subjected to SDS-PAGE using a 10% gel. The resolved proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Shanghai, China). The membrane was blocked with 5% skimmed milk or bovine serum albumin for 2 h at room temperature and incubated with primary antibodies overnight at 4°C. Next, the membrane was washed thrice with Tris-buffered saline with Tween 20 (TBST) and incubated with secondary antibodies for 2 h at room temperature. After washing thrice with TBST, the membrane was incubated with SuperSignal Chemiluminescent substrate (UltraSignal ECL Reagent, 4 A Biotech Co., Ltd, China). The protein bands were visualized using the Image Lab detection system (BioRad, Hercules, CA).
Image lab detection system
Manufactured by Bio-Rad
Sourced in United States
The Image Lab detection system is a versatile laboratory equipment designed for imaging and analyzing a variety of samples. It provides high-quality image capture and data analysis capabilities for diverse applications in the scientific community.
Automatically generated - may contain errors
Lab products found in correlation
Primescript 1st strand cdna synthesis kit, by Takara Bio (2 mentions)
Thermal cycler, by Bio-Rad (2 mentions)
Rnaiso, by Takara Bio (2 mentions)
Pcr mastermix 2x power taq, by BioTeke (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Rnase free ddh2o, by Takara Bio (1 mentions)
Protein a g agarose, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Proteintech (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab125219, by Abcam (1 mentions)
Anti sp1, by Proteintech (1 mentions)
O glcnac, by Abcam (1 mentions)
Anti p akt1, by Abcam (1 mentions)
Anti p 4e bp1, by Abcam (1 mentions)
Anti p p70s6k, by Abcam (1 mentions)
Anti p70s6k, by Abcam (1 mentions)
6 protocols using image lab detection system
1
Protein Extraction and Western Blotting
2
mRNA Extraction and Quantification from BMSCs
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Extraction of mRNA from BMSC was performed using RNAiso (Takara Bio, Japan) as explained in a previous article [45 (link)]. PrimeScript™ 1st strand cDNA Synthesis Kit (Takara Bio, Japan) was used to produce cDNA using 1μg of extracted mRNA. Primers shown in Table 2 were used in PCR amplification and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. PCR MasterMix 2X Power Taq (Bio Teke Corporation, China), cDNA, RNase Free ddH2O, and primers (Takara Bio, Japan) were mixed well in micro-tubes according to the manufacturer’s protocol and then loaded in the thermal cycler (Bio-Rad, USA), each cycle consisted of 30s for denaturation at 95 °C, 30s of annealing at 56.0, 56.5, 57, 57.5, or 58.0 °C, and 30s for extension at 72 °C, for a total of 35 or 40 cycles. The PCR outputs of all genes were dispensed in 2% agarose gels wells for electrophoresis in tris acetic acid EDTA (TAE) buffer. PCR bands were quantified by Image Lab detection system (Bio-Rad, USA) and Image J (ImageJ2x, Rawak Software Inc., Germany).
3
Immunoprecipitation and Immunoblotting of O-GlcNAcylated Proteins
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Proteins were extracted on ice with radioimmunoprecipitation buffer from 1 to 5 × 106 cells (Transgene, Beijing, China). For IP, lysates were incubated with the appropriate antibody: anti-SP1 (Proteintech, Wuhan, China). Subsequently, Protein A/G –agarose (Santa Cruz Biotechnology. Inc, USA) was added to lysates for 4 h to IP the target protein. For immunoblotting, protein samples or the IP were separated by 10% SDS-PAGE and transferred to nitrocellulose membranes (Millipore, Burlington, USA). The membranes were incubated with the following antibodies: O-GlcNAc (1:1000, ab2739, RL2, Abcam, Cambridge, MA, USA), OGT (1:1000, 11576–2-AP, Proteintech, Wuhan, China), OGA (1:1000, 14711–1-AP, Proteintech), AQP3 (1:1000, ab125219, Abcam), beta-actin (1:1000, 20536–1-AP, Proteintech), GLUT1 (1:1000, 66290–1-Ig, Proteintech), c-Myc (1:2000, 10828–1-AP, Proteintech), SP1 (1:2000, 21962–1-AP, Proteintech), Lamin B1 (1:2000, 12987–1-AP, Proteintech), at 4 °C overnight, respectively. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (Proteintech) for 1 h at room temperature and visualized using a chemiluminescent substrate (Invitrogen, USA) by an Image Lab detection system (Bio-Rad, USA).
4
Proteomic Analysis of BMSC Proteins
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Proteins were extracted from more than 106 harvested BMSCs according to the kit protocol (KeyGen Biotech, China). [46 (link)] BMSCs were digested in a lysis buffer cocktail and centrifuged at 12000 g for 15 minutes at 4 °C to get the supernatant as the total protein. 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) was used for electrophoresis of protein (20 μg) with loading buffer and then blotted to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., USA). Membranes were blocked with 5% skimmed milk in Tris-buffered saline tween 20, TBST, and then incubated overnight at 4°C with primary antibodies; anti-IHH, anti-P53, anti-P16, anti-PI3K, anti-p-PI3K, anti-Akt 1, anti-p-Akt 1, anti-NF-κB, anti-p-NF-κB-p65, anti-STAT3, anti-p-STAT3, anti-4EBP1, anti-p- 4EBP1, anti-p70S6K, anti-p-p70S6K and anti-β-actin (1:1000), all from Abcam, USA. The membranes were incubated for 1hour at RT with secondary antibody conjugated with horseradish peroxidase. Finally, TBST-washed membranes were treated with enhanced chemiluminescent (ECL) for detection (Bio-Rad, USA). Image Lab detection system (Bio-Rad, USA) was used for protein bands imaging and analysis.
5
Western Blot Analysis of Protein Extraction
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More than 106 harvested BMSCs were digested in a lysis buffer cocktail and centrifuged at 10000 xg for 15 minutes at 4℃ to get the supernatant as the total protein according to the kit protocol (KeyGen Biotech, China). 20 μg of extracted protein with a loading buffer were loaded in wells of 10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) for electrophoresis, and then blotted to a polyvinylidene fluoride (PVDF) membrane (Millipore Co., USA). Membranes were incubated in blocking buffer (5% skimmed milk in tris-buffered saline tween 20, TBST,) and then with indicated primary antibodies overnight at 4℃. After washing the primary antibodies, the membranes were incubated for 1h at RT° with secondary antibody conjugated with horseradish peroxidase. After TBST- washing, the membranes were treated with enhanced chemiluminescent (ECL) reagents (Bio-Rad, USA) for imaging. Image Lab detection system (Bio-Rad, USA) was used for protein bands imaging and analysis.
6
mRNA Extraction and Gene Expression Analysis
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RNAiso (Takara Bio, Japan) was used to extract mRNA of BMSCs, H9 human T lymphocytes, or FLSs as described in the previous article (26 (link)). cDNA was prepared using PrimeScriptTM 1st strand cDNA Synthesis Kit (Takara Bio, Japan) and 1 μg of extracted mRNA. The internal control was Glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Primers (Takara Bio, Japan) shown in Table 1 were used to amplify genes after being mixed with PCR MasterMix 2X Power Taq (Bio Teke Corporation, China), cDNA, RNase Free dH2O in micro-tubes according to the manufacturer’s instructions, and then incubated in the thermal cycler (Bio-Rad, USA). Each cycle consisted of 30s for denaturation at 95℃, 30s of annealing at 56.0, 56.5, 57, 57.5, or 58.0℃, and 30s for extension at 72℃, for a total of 35 cycles. Electrophoresis in tris acetic acid EDTA (TAE) buffer using 2% agarose gel was applied to separate and PCR products. Image Lab detection system (Bio-Rad, USA) or Image J (ImageJ2x, Rawak Software Inc., Germany) were used to quantify PCR bands.
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