Cells were washed twice in PBS and lysed in RIPA buffer (Solarbio, China) with 1 mM phenylmethanesulfonyl fluoride (PMSF). A BCA protein kit was used to quantify protein concentrations (Beyotime, China). Total proteins (30 μg) were separated on 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime, China). The membranes were then blocked in TBST with 3% non-fat milk (Sangon Biotech, China) at 25°C for 2 h and incubated with anti-survivin (1:1,000), anti-caspase-3 (1:1,000), anti-Bcl-2 (1:3,000), anti-P-gp (1:3,000), and anti-β-actin (1:5,000) primary antibodies (Proteintech, USA) overnight at 4°C. Next, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000) (Proteintech, USA) at 25°C for 1 h. Protein bands were visualized by ECL chemiluminescence kit (Sangon Biotech, China), and the gray values of the protein bands were analyzed by Image J.
Ecl chemiluminescence kit
Manufactured by Sangon
Sourced in China
The ECL chemiluminescence kit is a laboratory equipment designed to detect and quantify proteins in Western blot analysis. It utilizes a luminescent chemical reaction to generate a light signal proportional to the amount of target protein present in the sample. The kit includes all the necessary reagents and components to perform the chemiluminescence-based detection process.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Sangon (4 mentions)
Sab5500001, by Merck Group (4 mentions)
Rabbit human, by Merck Group (4 mentions)
Pvdf membrane, by Sangon (4 mentions)
Rbbp4, by Sangon (4 mentions)
Gadph, by Sangon (4 mentions)
D220395 rabbit human, by Sangon (3 mentions)
Ripa lysis system, by Sangon (3 mentions)
Twist, by Abcam (3 mentions)
Vimentin, by Abcam (3 mentions)
Pvdf membrane, by Beyotime (2 mentions)
Anti caspase 3, by Proteintech (1 mentions)
Anti bcl 2, by Proteintech (1 mentions)
Anti survivin, by Proteintech (1 mentions)
Anti p gp, by Proteintech (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Ripa buffer, by Solarbio (1 mentions)
Bca protein kit, by Beyotime (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Pvdf membrane, by Proteintech (1 mentions)
11 protocols using ecl chemiluminescence kit
1
Western Blot Analysis of Apoptosis Markers
2
Quantitative Analysis of CDK2 and β-Actin Proteins
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Quantitative analysis was performed after all the protein was extracted with RIPA lysis buffer (C500005, Sangon; Shanghai, China) from Hela and C-33A cells in different groups. An equal amount of protein was separated by 10% SDS-PAGE. The gel was immersed in a transfer buffer to achieve equilibrium before transferring it to a polyvinylidene fluoride membrane. Primary antibodies were diluted at a ratio of 1:1000. The membrane was later incubated for 2 h with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China). Following that, the hybrid membrane was blocked with 5% skimmed milk and incubated at 4 °C overnight. Next, the membrane was incubated for 2 h with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect proteins according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
3
6xHis-tagged Protein Detection by Western Blotting
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Western blotting was performed according to the method of X. Zhou et al. (2021) [21 (link)]. The purified peptide was separated by SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was washed with PBS-Tween 20 (PBST, 0.1% Tween 20) buffer for four times and incubated with anti-6×His tag mouse monoclonal antibody (Zoonbio Biotechnology, Nanjing, China) diluted by 5% skim milk blocking buffer (1:1000) at 4 °C overnight. After washing with PBST buffer three times, the membrane was incubated with a second antibody (goat anti-mouse IgG-conjugated alkaline phosphatase) prepared in 5% skim milk blocking buffer (1:10,000) at 37 °C for 1 h. Then the membrane was washed with PBST buffer four times. The positive bands on the membrane were detected using an ultrasensitive ECL chemiluminescence kit (Sangon Biotech, Shanghai, China).
4
Western Blot Analysis of EMT Markers
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The total proteins were extracted using a RIPA lysis system (Cat#: C500005, Sangon, China). Next, the proteins were separated with 10% SDS-PAGE gel and then transferred to PVDF membranes (Cat#: F019532, Sangon, China). After blocking the membranes in 5% skim milk, the hybridization membrane was incubated at 4 °C overnight with primary antibodies RBBP4 (Cat#: D154089, Sangon, China), Twist (Cat#: ab49254, Abcam, UK), Slug (Cat#: ab51772, Abcam, UK), Vimentin (Cat#: ab92547, Abcam, UK), MMP-2 (Cat#: ab92536, Abcam, UK) and GADPH (Cat#: D190090, Sangon, China). The membranes were then incubated with the corresponding secondary antibody for 2 h. Subsequently, the protein was detected using the ECL chemiluminescence kit (Cat#: C510043, Sangon, China) according to the manufacturer’s instructions.
5
Western Blot Analysis of MEG Antigen
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Western blotting was used to test the expression of MEG antigen in L-02 cells. Proteins (50 μg) were separated by 12% SDS-PAGE electrophoresis and the band on the gel was electro-transferred onto the pretreated PVDF membranes (Beyotime Biotechnology, China). Subsequently, the PVDF membranes were blocked with 5% non-fat milk in Tris-buffer saline with Tween 20 for 1 h at 25 °C, and then incubated with anti-His tagged antibodies at 4 °C overnight and horseradish peroxidase (HRP)-labeled goat anti-mouse secondary antibody (Proteintech, USA) for 1 h at 25 °C. Protein bands were visualized with the ultrasensitive ECL chemiluminescence kit (Sangon Biotech, China). The gray values of the protein bands were analyzed by Image J.
6
Quantitative Analysis of CDK2 and β-Actin Proteins
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Quantitative analysis was performed after all the protein was extracted with RIPA lysis buffer (C500005, Sangon; Shanghai, China) from Hela and C-33A cells in different groups. An equal amount of protein was separated by 10% SDS-PAGE. The gel was immersed in a transfer buffer to achieve equilibrium before transferring it to a polyvinylidene uoride membrane. Primary antibodies were diluted at a ratio of 1:1000.
The membrane was later incubated for 2 hours with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China). Following that, the hybrid membrane was blocked with 5% skimmed milk and incubated at 4 °C overnight. Next, the membrane was incubated for 2 hours with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scienti c, Inc., Waltham, MA, USA). Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect proteins according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
The membrane was later incubated for 2 hours with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China). Following that, the hybrid membrane was blocked with 5% skimmed milk and incubated at 4 °C overnight. Next, the membrane was incubated for 2 hours with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scienti c, Inc., Waltham, MA, USA). Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect proteins according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
7
Quantitative Protein Analysis of HeLa and C-33A Cells
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Quantitative analysis was performed after total protein was extracted with RIPA lysis buffer (C500005, Sangon; Shanghai, China) from Hela and C-33A cells in different groups. An equal amount of protein was separated by 10% SDS-PAGE. The gel was immersed in a transfer buffer for equilibrium, and then transferred to a polyvinylidene uoride membrane. Primary antibodies were diluted at a ratio of 1:1000.
The membrane was incubated with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China) for 2 hours.
The hybrid membrane was then blocked with 5% skimmed milk and incubated at 4 °C overnight. The membrane was subsequently incubated with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scienti c, Inc., Waltham, MA, USA) for 2 hours. Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect protein according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
The membrane was incubated with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China) for 2 hours.
The hybrid membrane was then blocked with 5% skimmed milk and incubated at 4 °C overnight. The membrane was subsequently incubated with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scienti c, Inc., Waltham, MA, USA) for 2 hours. Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect protein according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
8
Quantitative Protein Analysis of CDK2 and β-actin
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Quantitative analysis was performed after total protein was extracted with RIPA lysis buffer (C500005, Sangon; Shanghai, China) from Hela and C-33A cells in different groups. An equal amount of protein was separated by 10% SDS-PAGE. The gel was immersed in a transfer buffer for equilibrium, and then transferred to a polyvinylidene uoride membrane. Primary antibodies were diluted at a ratio of 1:1000. The membrane was incubated with diluted primary antibodies against CDK2 (D220395; Rabbit-Human; Sangon; Shanghai, China) and β-actin (SAB5500001; Rabbit-Human; Sigma-Aldrich, China) for 2 hours.
The hybrid membrane was then blocked with 5% skimmed milk and incubated at 4 °C overnight. The membrane was subsequently incubated with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scienti c, Inc., Waltham, MA, USA) for 2 hours. Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect protein according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
The hybrid membrane was then blocked with 5% skimmed milk and incubated at 4 °C overnight. The membrane was subsequently incubated with diluted secondary antibodies (A32731; Goat-Rabbit; Thermo Fisher Scienti c, Inc., Waltham, MA, USA) for 2 hours. Finally, a hypersensitive ECL chemiluminescence kit (C510043; Sangon; Shanghai, China) was used to detect protein according to the reagent instructions. The intensity of the protein bands was read using ImageJ software.
9
Protein Extraction and Western Blot Analysis
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The total proteins were extracted using a RIPA lysis system (Cat#: C500005, Sangon, China). Next, the proteins were separated with 10% SDS-PAGE gel and then transferred to PVDF membranes (Cat#: F019532, Sangon, China). After blocking the membranes in 5% skim milk, the hybridization membrane was incubated at 4 °C overnight with primary antibodies RBBP4 (Cat#: D154089, Sangon, China), Twist (Cat#: ab49254, Abcam, UK), Slug (Cat#: ab51772, Abcam, UK), Vimentin (Cat#: ab92547, Abcam, UK), MMP-2 (Cat#: ab92536, Abcam, UK) and GADPH (Cat#: D190090, Sangon, China). The membranes were then incubated with the corresponding secondary antibody for 2 hours. Subsequently, the protein was detected using the ECL chemiluminescence kit (Cat#: C510043, Sangon, China) according to the manufacturer's instructions.
10
Protein Extraction and Western Blotting
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Total proteins were extracted by RIPA lysis (Cat#: C500005, Sangon, China). Proteins were separated by 10% SDS-PAGE gel electrophoresis and transferred to PVDF membranes (Cat#: F019532, Sangon, China). After blocking the membranes in 5% skim milk, the hybridization membrane was incubated with primary antibodies RBBP4 (Cat#: D154089, Sangon, China) and GADPH (Cat#: D190090, Sangon, China) at 4 °C overnight. The membranes were continued to incubate with the corresponding secondary antibody for 2 hours, and the protein was detected using ECL chemiluminescence kit (Cat#: C510043, Sangon, China) according to the reagent instructions.
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