The membrane and cytosolic proteins were extracted from the isolated cardiomyocytes. The protein concentration was determined using bovine serum albumin (BSA) as the standard using a UV-2450 spectrophotometer (Shimadzu, Kyoto, Japan). Proteins subjected to heat denaturation were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. Subsequently, the membranes were blocked and incubated with a primary antibody, followed by incubation with the alkaline phosphatase-conjugated secondary antibody with BCIP/NBT chromogenic substrate. All images were captured and analyzed using the Gene Genius system (Syngene, Cambridge, UK).
Gene genius system
Manufactured by Syngene
Sourced in United Kingdom
The Gene Genius system is a laboratory equipment used for imaging and analyzing nucleic acids, such as DNA and RNA. It provides a platform for capturing and analyzing gel-based images, including those from agarose gels and polyacrylamide gels. The system utilizes digital imaging technology to capture and process the data.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer system, by Santa Cruz Biotechnology (3 mentions)
Hrp conjugated secondary antibody, by Abcam (2 mentions)
Ecl kit, by Thermo Fisher Scientific (2 mentions)
Uv 2450 spectrophotometer, by Shimadzu (1 mentions)
Total protein extraction kit, by Beyotime (1 mentions)
Quickblock blocking buffer, by Beyotime (1 mentions)
β actin, by Abcam (1 mentions)
Dnmt3b, by Abcam (1 mentions)
Bca kit, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Blocking buffer, by Beyotime (1 mentions)
Corresponding secondary antibody, by Abcam (1 mentions)
Caspase 12, by Abcam (1 mentions)
Blocking buffer, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Grp78, by Abcam (1 mentions)
6 protocols using gene genius system
1
Protein Extraction and Analysis in Cardiomyocytes
2
Protein Expression Analysis in Cardiac Tissue
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Isolated myocytes and minced ventricular tissue were homogenized and lysed by RIPA lysis buffer system (Santa Cruz). Protein was extracted by using Total Protein Extraction kit (Beyotime) according to manufacturer's instructions. A BCA kit (Pierce) was used to determine the concentration of protein. Protein was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated protein was electronically transferred to PVDF membranes. Membranes were incubated with QuickBlock™ blocking buffer (Beyotime) to eliminate the non‐specific bindings. Specific antibodies against SLN (1:1000, Abcam), SERCA2a (1:1000, Abcam), and β‐actin (1:5000, Abcam) were used to incubate the membranes at 4°C for 10 h and then washed by TBST for five and six times. HRP‐conjugated secondary antibodies (1:50 000, Abcam) was used to incubate the membranes at 37°C for 2 h and then washed by TBST for five and six times. ECL kit (Pierce) was used to develop the membranes, and the immunoblots were visualized with Gene Genius System (Syngene).
3
Quantification of Regulatory T Cell Proteins
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Isolated human CD4+CD25+ Tregs from peripheral blood and mouse CD4+CD25+ Tregs from spleen tissue were homogenized and lysed by RIPA lysis buffer system (Santa Cruz). Total protein was quantified with a BCA kit (Invitrogen). 40 μg protein sample was subjected to SDS-PAGE. Separated protein was then blotted onto PVDF membranes, which were then blocked with blocking buffer (Beyotime) at room temperature for 2 h before incubation with specific antibodies (DNMT3b, 1 : 2000, Abcam; FOXP3, 1 : 1000, Abcam; GAPDH, 1 : 1000, Abcam). The membranes were incubated at 4°C for 10 h and then washed by TBST for five or six times. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies (1 : 50000, Abcam) at 37°C for 2 h and then washed by TBST for five or six times. The membranes were developed by ECL kit (Thermo) and were detected by Gene Genius System (Syngene). The band intensities were measured using BandScan software.
4
Enzymatic Genotyping Optimization
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Enzyme digestion reactions were performed in a 20-mL reaction system containing 12.5 mL sterile distilled water, 5 mL PCR products, 2 mL 10X buffer, and 0.5 mL BstUI enzyme. The mixtures were incubated at 60°C for 4 h. Digestion products were analyzed by electrophoresis (1 h, 80 mA) on a 3% agarose gel. Photographic images were acquired using the Gene Genius system (Syngene International, Karnataka, India) to analyze the genotypes of KK, EK, and EE.
5
Western Blotting Analysis of UPR Markers
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Cells were collected and suspended in a RIPA lysis buffer system (Santa Cruz) and whole-cell extract was prepared. Total protein was extracted using the Protein Extraction kit (Beyotime) according to the instructions provided by the manufacturer. A BCA kit (Beyotime) was used to measure the protein concentrations. The protein was separated by vertical SDS-PAGE and then transferred to the PVDF membranes electronically. The blocking buffer (Santa Cruz) was applied to the membranes. Primary antibodies against GRP78 (1: 2000; Abcam), PERK (1: 2000; CST), p-PERK (1: 2000; CST), cleaved ATF6 (1: 2000; Abcam), IRE-1α (1: 4000; CST), p-IRE-1α (1: 2000; CST), CHOP (1: 2000; Abcam), caspase12 (1: 2000; Abcam), and GAPDH (1: 4000; Sigma) were used to incubate the membranes at 4°C for 8 h in TBST. Then, the corresponding secondary antibodies (Abcam) were used to incubate the membranes at room temperature for 1 h. The membranes were developed by an ECL kit (pierce) and then exposed with the Gene Genius system (Syngene). The densities of the blots were then analyzed with Image J software.
6
Protein Expression Analysis in Cardiomyocytes
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The proteins were extracted from cultured cardiomyocytes, using the Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA) to quantify protein concentrations as previously described [22] (link). Protein was then used for Western blotting with primary antibodies against Bax, Bcl-2, Caspase-3, Foxh1, c-Myc, p-Smad3 and APJ. All images were captured and analyzed by the Gene Genius system (Syngene, Cambridge, United Kingdom).
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