Proteins collected from myocardial tissues and H9C2 cells were lysed by RIPA lysate (Beijing Applygen Technolog Inc., China) according to the manufacturer's instruction. The protein content was quantified using the BCA method. After addition of loading buffer and boiling for 5 minutes at 99°C, protein samples (25 mg per sample) were separated by 10% Tris/Glycine SDS-PAGE and then electrotransferred to PVDF membranes at 300 mA for 1.5 h. Membranes were incubated in TBST containing 5% nonfat milk for 1 hours. Then the membranes were incubated with different primary antibodies including rabbit monoclonal antibodies against MFN1/2 (Proteintech), OPA1 (Proteintech), FIS1 (Proteintech), DRP1 (Proteintech), PGC-1α (Proteintech), NRF1(Proteintech), TFAM (Proteintech), and GAPDH (Proteintech) overnight at 4°C. The blots were washed with TBST three times and then incubated with anti-rabbit IgG (1 : 2000) for 4 h at 4°C. After washing three times with TBST, the immunolabeling was detected using Omni ECL reagent (EpiZyme, China) for 1 min at room temperature. The ultimate expression of every single protein was standardized by GAPDH. The band grayscale analysis was performed using Image J software.
Omni ecl reagent
Manufactured by Ipsen
Sourced in China
The Omni ECL reagent is a chemiluminescent detection solution used in Western blotting and other immunoassay techniques to visualize and quantify target proteins. The reagent produces a luminescent signal when it reacts with the horseradish peroxidase (HRP) label on the secondary antibody, enabling the detection of the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Rcl1 15330 1 ap, by Proteintech (2 mentions)
Bs 40295g hrp, by Bioss Antibodies (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Pgc 1α, by Proteintech (1 mentions)
Mfn1 2, by Proteintech (1 mentions)
Gapdh, by Proteintech (1 mentions)
Gapdh bs 0755r, by Bioss Antibodies (1 mentions)
Pierce bca, by Thermo Fisher Scientific (1 mentions)
Ps108, by Ipsen (1 mentions)
Af5186, by Affinity Biosciences (1 mentions)
Flag 80010 1 rr, by Proteintech (1 mentions)
Ha 51064 2 ap, by Proteintech (1 mentions)
Pdl 1 13 684, by Cell Signaling Technology (1 mentions)
Fd006, by Fdbio (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Gapdh 60004 1 ig, by Proteintech (1 mentions)
Procaspase 3, by Cell Signaling Technology (1 mentions)
Chemiscope 6200 system, by Clinx (1 mentions)
10 protocols using omni ecl reagent
1
Mitochondrial Dynamics Protein Analysis
2
Western Blot Analysis of Protein Targets
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Cells were lysed in RIPA buffer (Beyotime Biotechnology, China) containing 1X Protease and Phosphatase inhibitor (Beyotime Biotechnology, China). An equal amount of protein samples was separated by 8% SDS/PAGE and transferred to 0.25 mm polyvinylidene fluoride membranes (Millpore, Germany). The membranes were blocked with 5% non-fat milk for 1 h, then incubated with the individual antibody at 4 ℃ overnight: Tubulin (YFB6011, YIFAN BIOLOGICAL), Rcl1 (15330-1-AP; Proteintech), GAPDH (bs-0755R, Bioss). The membranes were then incubated with the second antibody (bs-40295G-HRP, Bioss) at 37 ℃ for 1 h. Finally, protein bands were visualized using Omni ECL reagent (EpiZyme, China), and the gray intensity was acquired by using Fiji (NCBI, USA).
3
Western Blot Analysis of Bone and Apoptosis Markers
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The protein was extracted from the cells using radio immunoprecipitation assay (PC101, RIPA) lysis buffer (EpiZyme, Shanghai, China). The protein concentration was determined using Thermo Scientific™ Pierce™ BCA (23227). An equal amount of protein was separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with protein-free fast blocking buffer (PS108, EpiZyme), it was incubated with primary antibodies against Runx2 (Affinity, AF5186), Bcl-2 (Affinity, AF6139), Caspase-3 (Huabio, ET1608–64), and GAPDH (EpiZyme, LF205) at 4 °C overnight, and then incubated with the secondary antibody at 37 °C for 1 h. Thereafter, the proteins were visualized using Omni ECL reagent (SQ201, ECL; EpiZyme) under e-Blot (Touch Imager, Shanghai, China). The gray densitometric was analyzed using Image J (USA).
4
Dot Blot Immunoassay for S. agalactiae
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Dot blot assays were performed as previously described, with some modifications (55 (link)). Briefly, overnight cultures were inoculated into 10 mL THB at a ratio of 1:100, washed twice with PBS, and adjusted to 1 × 1010 CFU/mL. Aliquots (5 μL) of bacteria serially diluted 2-fold were spotted onto nitrocellulose membranes. After being fixed with 70% ethanol for 5 min and air dried, the membrane was blocked with 5% (wt/vol) skim milk in PBST (PBS containing 0.05% Tween 20) at 4°C overnight. Then, the membrane was incubated with hyperimmune serum (1:500 dilution) against S. agalactiae GD201008-001 at 37°C for 1.5 h and washed three times with PBST. The membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000 dilution) at 37°C for 1.5 h and washed three times with PBST. Finally, the reaction was visualized with Omni ECL reagent (EpiZyme, Shanghai, China) under a ChemiDoc touch imaging system (Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis Protocol
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Western blot anlysis was performed as previously described (9). Briefly, samples were collected in Laemmli buffer, and total proteins were separated by SDS polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore). The membrane was blocked using 5% dried milk and incubated with the indicated primary antibody and secondary antibody. Bands were visualized using Omni ECL reagent (EpiZyme) under GE AI600, and the gray intensity was acquired by using Fiji (NCBI).
6
Protein Expression Analysis with RIPA Lysis
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The radio immunoprecipitation assay lysis buffer (RIPA) (P0013B, Beyotime, China) containing a protease-inhibitor cocktail (HY-K0010, MCE, USA) were used to extract proteins from cells. The proteins were lysed in SDS-loading buffer (FD006, Fdbio, China), then 20μg proteins were resolved on a 10% sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane (IPVH00010, Millipore, USA). The membrane was incubated with primary antibodies against PD-L1(13684, Cell Signaling Technology, USA), FLAG (80010-1-RR, Proteintech, USA), STT3A (12034-1-AP, Proteintech, USA), c-Jun (AF6089, Affinity, USA), HA (51064-2-AP, Proteintech, USA) and GAPDH (60004-1-Ig, Proteintech, USA) at a dilution of 1:1000, followed by incubation with species-specific (rabbit or mouse) HRP-conjugated secondary antibodies at a dilution of 1:5000. The immunoreactive bands were visualized by Omni ECL reagent (SQ101, EpiZyme, China).
7
Dot Blot Immunoassay for S. agalactiae
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Dot blot assays were performed as previously described, with some modifications (55 (link)). Briefly, overnight cultures were inoculated into 10 mL THB at a ratio of 1:100, washed twice with PBS, and adjusted to 1 × 1010 CFU/mL. Aliquots (5 μL) of bacteria serially diluted 2-fold were spotted onto nitrocellulose membranes. After being fixed with 70% ethanol for 5 min and air dried, the membrane was blocked with 5% (wt/vol) skim milk in PBST (PBS containing 0.05% Tween 20) at 4°C overnight. Then, the membrane was incubated with hyperimmune serum (1:500 dilution) against S. agalactiae GD201008-001 at 37°C for 1.5 h and washed three times with PBST. The membrane was incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:10,000 dilution) at 37°C for 1.5 h and washed three times with PBST. Finally, the reaction was visualized with Omni ECL reagent (EpiZyme, Shanghai, China) under a ChemiDoc touch imaging system (Bio-Rad, Hercules, CA, USA).
8
Western Blot Analysis of Protein Expression
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Protein lysates from isolated tissues were extracted using RIPA buffer with protease inhibitors. The protein concentration was measured with a BCA assay, and an equal amount of protein was separated by SDS PAGE electrophoresis and transferred to PVDF membranes. Membranes were blocked with 5% milk powder in Tris-buffered saline-tween 20 (TBST) for 1 h and then incubated with specific primary antibody (in blocking solution) at 4°C overnight. Next, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 h at RT. After several washes with TBST, the membrane was incubated with Omni ECL reagent (EpiZyme) and imaged by a ChemiScope 6200 system (CLINX). The following primary antibodies were used for the experiments. Antibodies against β-actin (1:5,000, A5441; Sigma-Aldrich), β-Tubulin (1:5,000, #5346; Cell Signaling), CHOP (1:1,000, #2895; Cell Signaling), Procaspase-3 (1:1,000, #9662; Cell Signaling), Cleaved Caspase-3 (1:1,000, #9664; Cell Signaling), BAX (1:1,000, #2772; Cell Signaling), and BCL2 (1:1,000, #3498; Cell Signaling) were used.
9
TRIB3 Protein Expression Analysis by Western Blot
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Western blot was used to assess TRIB3 protein expression as previously described (18 (link)). Briefly, whole cell lysates were prepared and quantified using RIPA reagent. Protein samples were separated using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes (Bio-Rad). Membranes were incubated in a blocking solution, followed by incubation with rabbit anti human monoclonal antibody against TRIB3 (1:1,000, ab137526, Abcam, USA), SMARCD3 (SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily D, member 3; 1:1,000, ab171075,Abcam, USA), or glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000, Abcam, USA) for 18 hours at 4 °C. Membranes were then washed three times with Tris-buffered saline Tween (TBST) and incubated with a goat anti-rabbit IgG-AP secondary antibody (1:5,000 dilution, #HA1019, HuaBio, Hangzhou, China) in room temperature for 1 hour. After washing, the proteins on the membrane were detected by an enzyme labeling method and chemiluminescence in accordance with the manufacturer’s instructions (Omni ECL reagent (EpiZyme, Shanghai, China).
10
Western Blot Analysis of Protein Expression
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Cells were lysed in RIPA buffer (Beyotime Biotechnology, China) containing 1X Protease and Phosphatase inhibitor (Beyotime Biotechnology, China). An equal amount of protein samples was separated by 8% SDS/PAGE and transferred to 0.25mm polyvinylidene uoride membranes (Millpore, Germany). The membranes were blocked with 5% non-fat milk for 1h, then incubated with the individual antibody at 4℃ overnight: Tubulin (YFB6011, YIFAN BIOLOGICAL), Rcl1 (15330-1-AP, Proteintech), GAPDH (bs-0755R, Bioss). The membranes were then incubated with the second antibody (bs-40295G-HRP, Bioss) at 37℃ for 1h. Finally, protein bands were visualized using Omni ECL reagent (EpiZyme, China), and the gray intensity was acquired by using Fiji (NCBI, USA).
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