After imaging, brain tissues were lysed in ice-cold radioimmunoprecipitation assay buffer and total protein lysates were fractionated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were probed with antibodies against iba-1 (GTX100042) from GeneTex Biotechnology (Irvine, CA) at 1/1000 dilution, and TSPO (NB100-41398) from Novus Biologicals (Centennial, CO, USA) at 1/10,000 dilution and β-actin (NB600-501) from Novus Biologicals (Centennial, CO, USA) at 1/1000 dilution. After washing three times for 10 min in Tris-buffered saline supplemented with 0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, washed with TBST and the signals were visualized and quantified using the GeneGnome Chemiluminescence Imaging System (Syngene, Frederick, MD, USA).
Genegnome chemiluminescence imaging system
Manufactured by Syngene
Sourced in United States, United Kingdom
The GeneGnome chemiluminescence imaging system is a lab equipment designed for the detection and analysis of chemiluminescent signals. It is used for imaging and quantifying protein and nucleic acid samples in various applications such as Western blotting, Northern blotting, and in-vitro diagnostics.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
β actin nb600 501, by Novus Biologicals (1 mentions)
Owl hep 1 semidry electroblotting system, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Bio-Rad (1 mentions)
Image studio lite 4, by LI COR (1 mentions)
Clarity western ecl blotting substrate, by Bio-Rad (1 mentions)
Anti tet1, by Abcam (1 mentions)
Anti tet2, by Active Motif (1 mentions)
Anti gfp, by Thermo Fisher Scientific (1 mentions)
4 protocols using genegnome chemiluminescence imaging system
1
Western Blot Analysis of Neuroinflammatory Markers
2
Immunoblotting for Protein Detection
3
HUVEC Protein Expression Analysis
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Total protein of the HUVECs was obtained as previously described (15 (link)). The protein expression of PECAM-1, FoxO1, phospho-FoxO1, Akt, and phospho-Akt in HUVECs were analyzed using western blotting. SDS-PAGE (10% gel) was used to separate an equal quantity (40 µg) of proteins, which were then transferred onto PVDF membranes. After blocking with 5% BSA (cat. no. SBJ-DB2040; Nanjing SenBeiJia Biological Technology Co., Ltd.) for 2 h at 25°C, incubation of the membranes was performed at 4°C with anti-PECAM-1 (1:1,000), anti-FoxO1 (1:1,000), anti-phospho-FoxO1 (1:1,000), anti-Akt (1:1,000), anti-phospho-Akt Ser473 (1:1,000), anti-phospho-Akt Thr308 (1:1,000) and anti-β-actin (1:1,000) antibodies overnight. The membranes were washed with TBS with 0.1% Tween-20 (cat. no. T8220; Beijing Solarbio Science & Technology Co., Ltd.) and incubated with HRP-conjugated secondary antibodies (1:10,000) for 2 h at 25°C. The antibody-bound target antigens were detected using enhanced chemiluminescence reagents (cat. no. WBKLS0500; EMD Millipore). A GeneGnome chemiluminescence imaging system (Syngene) was used to capture images, and ImageJ software (v.1.8.0; National Institutes of Health) was used to analyze the target protein expression. The experiments were performed at least in triplicate.
4
Validating TET Protein Production in HEK293 Cells
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To verify whether transfection with pSELECT-GFPzeo-TET1 or pSELECT-GFPzeo-TET2 vectors results in the production of the respective TET proteins, HEK293 cells were suspended in 500 µL of RIPA buffer containing protease inhibitor mix, incubated on ice for 10 min, and then centrifuged at 400× g for 20 min at 4 °C. The protein concentration was determined using the BCA Protein Assay kit (Thermo Fisher Scientific, Waltham, MA, USA). Twenty micrograms of the extract were used for electrophoresis and transferred to the membrane following standard procedures. The membrane was blocked and incubated with anti-TET1 (1:1000; Abcam, Cambridge, UK), anti-TET2 (1:500, Active-Motif, Carlsbad, CA, USA), or anti-GFP (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) primary antibodies, washed in TBST and incubated with the appropriate secondary antibodies. Signals were detected using the Clarity ECL Western Blotting Substrate (Bio-Rad, Hercules, CA, USA) and analyzed using a GeneGnome chemiluminescence imaging system (Syngene, Cambridge, UK) and Image Studio Lite 4.0 software (LI-COR, Lincoln, NE, USA) (Supplementary Figure S1 ).
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