Proteins were extracted using RIPA lysis buffer (Thermo Scientific, IL, USA) supplemented with protease inhibitor (Roche, Mannheim, Germany) and phosphatase inhibitor (Thermo Scientific, IL, USA) cocktails. A BCA Protein Assay Kit (Beyotime Biotechnology, Shanghai, China) was used to assess the protein concentration. After the protein was denatured at 100 °C, a PAGE Gel Fast Preparation Kit (Epizyme, Shanghai, China) was applied to perform sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‒PAGE), and the proteins were transferred onto a PVDF membrane (Millipore, MA, USA) at 300 mA for 65 min. Afterward, the membranes were blocked for 60–90 min in 5% skim milk powder and incubated with primary antibodies overnight at 4 °C. After washing 3 times using 1 × TBST, the cells were incubated with secondary antibodies for 1 h. Immunoreactivity was visualized using enhanced chemiluminescent (ECL) chromogenic substrate (Millipore, MA, USA). The membranes were finally detected by using a ChemiDoc MP Imager System (Bio-Rad, CA, USA).
Ecl chromogenic substrate
Manufactured by Merck Group
Sourced in United States
ECL chromogenic substrate is a laboratory reagent used in immunoassays for the detection and quantification of proteins. It serves as a substrate for the enzyme-catalyzed oxidation reaction, resulting in the production of a colored product. The core function of this product is to provide a colorimetric signal that can be measured to determine the presence and amount of the target analyte.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Ripa reagent, by Solarbio (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Page gel fast preparation kit, by Ipsen (1 mentions)
Chemidoc mp imager system, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
C myc, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Abcam (1 mentions)
Bcl2 associated x (bax), by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Bca kit, by Beyotime (1 mentions)
Gapdh, by Proteintech (1 mentions)
Anti histone 3, by Abcam (1 mentions)
9 protocols using ecl chromogenic substrate
1
Western Blot Protein Analysis Protocol
2
Western Blot Analysis of Lin28A
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Cells were collected, washed with ice-cold PBS, lysed using RIPA reagent containing 1% PMSF, and centrifuged at 12,000×g for 5 min. The protein samples were separated on a 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to a 0.2 µm polyvinylidene fluoride membrane (Millipore, Billerica, MA, USA), which was then blocked with 5% milk for 2 h. The choice and dilution factor of antibodies used in western blotting were described as follows: primary antibodies—rabbit anti-Lin28A (1:500, 16177-1-AP, Proteintech, Chicago, IN, USA) and rabbit anti-GAPDH (1:2000, 10494-1-AP, Proteintech); secondary antibody-goat anti-rabbit horseradish peroxidase-labeled (1:20,000, ZB-2301, ZSJQ-bio, Beijing, China). The blocked membrane was first incubated with the primary antibodies of choice overnight, washed with TBST buffer, and then incubated with the secondary antibodies for 2 h. Protein–antibody complexed were visualized by ECL chromogenic substrate (Millipore) and quantified by densitometry using ImageJ (National Institutes of Health, USA) with GAPDH as control.
3
Quantitative Protein Analysis by Western Blot
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Total proteins were extracted using RIPA lysis buffer (Beyotime) and quantified using Pierce BCA Protein Assay Kit (Thermo Fisher). Proteins were separated by SDS-PAGE and transferred onto PVDF membrane. The membrane was blocked in defatted milk powder and incubated at 4 ℃ overnight in NR1 primary antibody (Cell Signaling Technology, 1:1000). Membranes were washed in PBST 5 min for 3 times and then incubated in secondary antibody (Bioss, 1:5000) for 1 h at room temperature. ECL chromogenic substrate (Millipore) was added for development and was imaged using a gel imaging system.
4
Western Blot Analysis of Protein Expression
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Proteins were extracted from cells with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime) and quantified using a BCA Protein Assay Kit (Beyotime). An equivalent amount of protein was loaded on 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The PVDF membranes were blocked with 5% nonfat milk for 1 h at 37°C and then incubated overnight at 4°C with diluted antibodies for c-Myc (1:1,000), p53 (1:1,000), Bcl-2 (1:500), Bax (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), or GAPDH (1:5,000; Abcam, Cambridge, MA, USA). After being washed three times with TBST buffer, membranes were incubated with secondary horseradish peroxidase–goat anti-rabbit/mouse antibodies (1:10,000; Santa Cruz Biotechnology). Specific bands were detected using enhanced chemiluminescence (ECL) chromogenic substrate (Millipore) following the manufacturer’s instructions. GAPDH was the control.
5
Western Blot Analysis of Cellular Proteins
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Total proteins of cells were extracted, quantified using BCA kit (23235, Thermo), separated by 10% SDS-PAGE, and transferred onto polyvinylidene difluoride membrane. The membrane was blocked with 5% defatted milk powder for 1.5 h, and was incubated overnight in primary antibody (anti-β-Actin at 1:10000; anti-MDK at 1:1000) at 4°C. On the following day, the membrane was incubated in secondary antibody (goat anti-rabbit IgG-HRP 1:20000) for 1 h at room temperature. After adding ECL chromogenic substrate (Millipore, US), the membrane was imaged using a gel imaging system.
6
Western Blot Analysis of VIRMA Protein
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Cell lysates were prepared using RIPA Lysis Buffer containing protease and phosphorylase inhibitors (Beyotime Biotechnology, China), and the BCA protein assay kit (Beyotime Biotechnology, China) was used for protein quantification. Samples were electrophoresed using 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22 µm polyvinylidene fluoride (PVDF) membranes (Millipore, Massachusetts, USA). After incubation with the primary antibodies, the membrane was incubated with the secondary antibody. Color development was performed using an enhanced chemiluminescence (ECL) chromogenic substrate (Millipore, Massachusetts, USA). The antibodies used in this study were as follows: VIRMA (1:500 dilution, #88358; Cell Signaling Technology, USA), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:1,000 dilution, #5174; Cell Signaling Technology, USA), and anti-rabbit IgG horse radish peroxidase (HRP)-linked antibody (1:2,000 dilution, #7074; Cell Signaling Technology, USA).
7
Quantifying KCNJ2 Protein in Rabbit Atria
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The protein levels of KCNJ2 in rabbit atria and cardiomyocytes were measured by Western blotting. The protein content of the lysates was determined using a BCA kit (Beyotime, Shanghai, China). Protein lysates were separated using 10% SDS‐PAGE and then transferred to a polyvinylidene fluoride membrane (Millipore, MA). After blocking with 5% skim milk powder for 1 hour, the membranes were incubated with primary antibodies specific for KCNJ2 (1:500, Proteintech, China) or GAPDH (1:1000, Proteintech, China) at 4°C overnight. The membranes were then washed and incubated with secondary antibody for 2 hours at room temperature. Quantification was performed using ECL chromogenic substrate (Millipore, MA) on a densitometer.
8
Protein Extraction and Western Blot Analysis
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Total protein was extracted and lysed in RIPA reagent (Solarbio) with 1% PMSF (Solarbio). Specifically, the nuclear protein was extracted using the Nuclear Protein Extraction Kit (Solarbio). Then, the lysates were separated on 10% or 15% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) following transfer to a polyvinylidene fluoride membrane (PVDF; Millipore). After blocking with 5% skimmed milk, these membranes were incubated with the following primary antibodies: anti-RUNX2 (Cell Signaling Technology), anti-ALP (Proteintech Group, Inc.), anti-LDHA (Abcam), anti-β actin (Abcam), anti-Histone 3 (Abcam), and anti-lactyl-lysine (PTM Bio) overnight at 4 °C. Following washing with Tris-buffered saline containing 0.05% Tween 20, the second antibody was added and incubated for 1 h. The visualization was performed with the ECL chromogenic substrate (Millipore).
9
Quantifying Osteoblastic Protein Expression
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PDLSCs were collected and lysed in RIPA reagent (Solarbio) containing 1% PMSF. After heating, the protein samples were separated in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and transferred to a 0.2 μm polyvinylidene uoride membrane (Millipore, Burlington, MA, USA). After blocking in 5% skimmed milk, membranes were incubated overnight at 4 ℃ with the following primary antibodies: rabbit anti-RUNX2 (1:1000, Lot # 8486S; Cell Signaling Technology, MA, USA), rabbit anti-ALP (1:1000, 11187-1-AP; Proteintech, Rosemont, IL, USA), rabbit anti-EZH2 (1:500, Lot # 2477979; Millipore), rabbit anti-embryonic ectoderm development (EED) (1:500, 16818-1-AP; Proteintech), rabbit antisuppressor of zeste 12 (SUZ12) (1:500, 20366-1-AP; Proteintech) and rabbit anti-GAPDH (1:2000, 10494-1-AP, Proteintech, USA). After washing in Tris-buffered saline solution (TBS-T, Solarbio) with Tween-20, the membranes were incubated at room temperature for 1 h with peroxidase-conjugated anti-rabbit IgG (SA00001-2; Proteintech). ECL chromogenic substrate (Millipore) was used to detect the immunoreactive bands, and ImageJ software (National Institutes of Health, Bethesda, USA) was used to quantify the densitometry using GAPDH as the control.
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