Cells were collected and lysed in RAPI buffer (Solarbio, Beijing, China) with protease/phosphatase inhibitor (Cell Signaling Technology, Canada). After treatment on ice for 30 min, lysates were clarified by centrifugation at 12000 rpm for 15 min at 4°C and the protein content was measured using a BCA protein assay kit (Beyotime, Jiangsu, China). Sample protein was mixed with 5× Loading Buffer (Beyotime, Jiangsu, China). The western blot was similar as described by Gao et al. [36 (link)]. In brief, the samples were separated by SDS-PAGE and electrotransferred onto a polyvinylidene-difluoride membrane (Pall Corporation, Mexico). The membrane was blocked with BSA blocking buffer for two hours at room temperature, incubated overnight at 4°C with interest primary antibodies (Santa Cruz Biotechnology, USA, or Cell Signaling Technology, Canada) in PBST. After washing, the membrane was incubated with an appropriate secondary antibody (Santa Cruz Biotechnology, USA) for 30 min. The membrane was incubated with streptavidin HRP (Thermo, USA) for 30 min after washing. The blotted protein bands were detected by Chemiluminescent Substrate kit (BIO-RAD, USA).
Chemiluminescent substrate kit
Manufactured by Bio-Rad
Sourced in United States
The Chemiluminescent substrate kit is a laboratory product designed for the detection and quantification of proteins in Western blot analysis. It provides a chemiluminescent reagent that produces a luminescent signal upon interaction with the target protein, allowing for the visualization and analysis of the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
Rapi buffer, by Solarbio (2 mentions)
Protease phosphatase inhibitor, by Cell Signaling Technology (2 mentions)
Loading buffer, by Beyotime (2 mentions)
Streptavidin hrp, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Pall Corporation (1 mentions)
Alkaline phosphatase conjugated anti rabbit igg, by Bio-Rad (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Western blocking reagent, by Roche (1 mentions)
Biodoc it imaging system, by Analytik Jena (1 mentions)
Ab97779, by Abcam (1 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Ab32503, by Abcam (1 mentions)
Ripa lysis buffer, by Elabscience (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab38898, by Abcam (1 mentions)
Ab32124, by Abcam (1 mentions)
Appropriate secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride pvdf membranes, by Pall Corporation (1 mentions)
5 protocols using chemiluminescent substrate kit
1
Western Blotting of Protein Lysates
2
SDS-PAGE and Western Blotting Analysis of Proteins
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Proteins were separated by 12.5% or 15% SDS-PAGE on a 0.1% SDS-Tris-glycine running buffer system and stained with Coomassie blue or silver using a silver staining kit (Daiichi Pure Chemical, Tokyo, Japan). For western blotting, SDS-PAGE gels were transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad) in a buffer containing 20% methanol, 25 mM Tris (pH 8.3), and 192 mM glycine. Membranes were incubated for 30 min in blocking buffer consisting of 1% Western Blocking Reagent (Roche Molecular Diagnostics, Indianapolis, IN, USA) in maleic acid buffer (100 mM maleic acid and 150 mM NaCl, adjusted to pH 7.5 with NaOH). The membranes were incubated with rabbit antiserum (1:1000 in maleic acid buffer), and then alkaline phosphatase-conjugated anti-rabbit IgG (Bio-Rad) diluted 1:10,000 in maleic acid buffer. Bands were visualized using a chemiluminescent substrate kit (Bio-Rad).
3
Western Blot Protein Analysis Protocol
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The concentration of extracted proteins was measured by the BCA method.35 (link) WB experiments were operated referring to the published paper.36 (link) Additionally, blocking was performed with 5% skim milk. β-actin was the loading control. After incubated with the primary antibodies (shown in Table 2 ) at 4 overnight and the following secondary antibodies at 26°C for 2 h, the protein bands were visualized using a chemiluminescent substrate kit (Bio-Rad, California, USA, NO.1705060) and photographed using BioDoc-it Imaging System (UVP, Upland, USA). Besides, the qualification of protein bands was conducted by Image J (National Institutes of Health, Bethesda, Maryland, USA).
Information on the primary antibodies.
| Name | Brand | No. | Dilution rate |
|---|---|---|---|
| β-actin | CST | #3700 | 1:1000 |
| N-cadherin | CST | #4061 | 1:1000 |
| E-cadherin | CST | #3195 | 1:1000 |
| Vimentin | CST | #3932 | 1:1000 |
| Snail | Abcam | ab216347 | 1:1000 |
| Slug | Abcam | ab302780 | 1:1000 |
| p-STAT3 | Abcam | ab267373 | 1:1000 |
| STAT3 | CST | #9139 | 1:1000 |
| p-AKT | CST | #4060 | 1:2000 |
| AKT | CST | #4060 | 1:2000 |
| p-smad2 | Abcam | ab280888 | 1:1000 |
| p-smad3 | Abcam | ab52903 | 1:2000 |
| smad2/3 | CST | #8685 | 1:1000 |
| β-catenin | CST | #8480 | 1:1000 |
4
Western Blot Analysis of Protein Expression
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Total proteins were isolated from cells using RIPA Lysis Buffer (Elabscience, Wuhan, China), and then quantified using a BCA Protein Assay Kit (ThermoFisher, SanJose, CA, USA). The protein samples were separated by 10% SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was then blocked in 5.0% non-fat milk for 45 min and incubated with primary antibody at 4°C overnight. The primary antibodies included anti-GAPDH (1:1000, ab9485, Abcam, Cambridge, England), -PI3K (1:1000, 4292, CST, Danvers, MA, USA), -p-PI3K (1:1000, 17,366, CST), -Akt (1:1000, 9272, CST), -p-Akt (1:1000, 4060, CST), -MMP-2 (1:1000, ab97779, Abcam), -MMP-9 (1:1000, ab38898, Abcam,), -Bcl-2 (1:1000, ab32124, Abcam), and -Bax (1:1000, ab32503, Abcam). Subsequently, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:10,000, G-21234, Invitrogen) for 1 h at 25°C. Protein bands were visualized using a Chemiluminescent Substrate Kit (Bio-Rad, Hercules, CA, USA). GAPDH was used as the internal control.
5
Western Blot Analysis of Protein Samples
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Samples were collected and lysed in RAPI buffer (Solarbio, Beijing, China) with a protease/phosphatase inhibitor (Cell Signaling Technology, Canada). After treatment on ice for 30 min, lysates centrifuged at 12,000 rpm for 15 min at 4°C, and the protein content was measured using a BCA protein assay kit (Beyotime, Jiangsu, China).
Protein samples were mixed with 5×loading buffer (Beyotime, Jiangsu, China). The samples were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation, Mexico). The membranes were blocked with BSA blocking buffer for two hours at room temperature, incubated overnight at 4°C with the primary antibodies (Santa Cruz Biotechnology, USA or Cell Signaling Technology, Canada) in PBST. After washing, the membranes were incubated with the appropriate secondary antibody (Santa Cruz Biotechnology, USA) for 30 min. The membranes were incubated with streptavidin HRP (Thermo, USA) for 30 min after washing. The blotted protein bands were detected using a chemiluminescent substrate kit (Bio-Rad, USA). The signal intensity of blotting was normalized to the Western signal of the corresponding total protein. Relative intensities of protein bands were analyzed by Image J software.
Protein samples were mixed with 5×loading buffer (Beyotime, Jiangsu, China). The samples were separated by SDS-PAGE and electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Pall Corporation, Mexico). The membranes were blocked with BSA blocking buffer for two hours at room temperature, incubated overnight at 4°C with the primary antibodies (Santa Cruz Biotechnology, USA or Cell Signaling Technology, Canada) in PBST. After washing, the membranes were incubated with the appropriate secondary antibody (Santa Cruz Biotechnology, USA) for 30 min. The membranes were incubated with streptavidin HRP (Thermo, USA) for 30 min after washing. The blotted protein bands were detected using a chemiluminescent substrate kit (Bio-Rad, USA). The signal intensity of blotting was normalized to the Western signal of the corresponding total protein. Relative intensities of protein bands were analyzed by Image J software.
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