The whole cell lysates were obtained from cells and protein concentrations were determined using the Bio-Rad (Hercules, CA, USA) protein assay. Afterwards, whole cell lysates were solubilized in 4× SDS sample buffer and separated on 10% SDS polyacrylamide gels. Membranes (Millipore, Billerica, MA, USA) were incubated with antibodies against ERK1/2, AMPKα, pERK1/2, p-AMPKα, Sp1 and DNMT1 (1:1000). The membranes were washed and incubated with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling, Beijing, China). The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millipore, Shanghai, China), followed by observing signals under the Gel Imagine System (Bio-Rad) for up to 1 min., or exposed to X-ray film.
Gel imagine system
Manufactured by Bio-Rad
Sourced in United States
The Gel Imagine System is a laboratory instrument designed for the visualization and documentation of electrophoresis gels. It provides a platform for capturing high-quality images of stained gels, which can be used for analysis and documentation purposes.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon western, by Merck Group (7 mentions)
Secondary antibody raised against rabbit igg conjugated to horseradish peroxidase, by Cell Signaling Technology (3 mentions)
Goat anti rabbit igg h l hrp, by Abcam (2 mentions)
Cleaved caspase 3, by Abcam (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
Gapdh, by Abcam (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protein assay, by Bio-Rad (1 mentions)
Ecl solution, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Secondary goat antibody raised against rabbit igg conjugated to horseradish peroxidase, by Cell Signaling Technology (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Ab109520, by Abcam (1 mentions)
Anti p21, by Abcam (1 mentions)
Anti dnmt1, by Abcam (1 mentions)
Anti sox5, by Merck Group (1 mentions)
Secondary antibody conjugated to horseradish peroxidase, by Cell Signaling Technology (1 mentions)
11 protocols using gel imagine system
1
Western Blot Analysis of Signaling Proteins
2
Immunoblotting Technique for Protein Analysis
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The detailed method was based on previous report [31 (link)]. The cell lysates containing same amount of protein were separated on SDS polyacrylamide gels. Membranes (Millipore, Billerica, MA, USA) were incubated with antibodies against the total and phosphor-form SAPK/JNK, p50, p65, EZH2 and DNMT1 (1:1000). The membranes were washed and incubated with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling, Beverly, MA, USA), followed by transferring to freshly made ECL solution (Immobilon Western; Millipore, Billerica, MA, USA), and documented the signals using the Gel Imagine System (Bio-Rad, Hercules, CA, USA).
3
Western Blot Analysis of Protein Expression
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The cell lysates containing equal amounts of protein were separated on SDS polyacrylamide gels. Membranes (Millipore, Billerica, MA, USA) were incubated with antibodies against c-Jun and p21 (1:1000), respectively. Afterward, following washing and incubating with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling Technology), the membranes then were transferred to fresh-made ECL solution (Immobilon Western; Millipore), and the signals were documented using the Gel Imagine System (Bio-Rad, Hercules, CA, USA). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to quantify and compare the intensity of single bands between the control and proteins of interest.
4
Western Blot Analysis of Protein Markers
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The detailed method was based on previous report 33 . Whole cell lysates containing same amount of protein were solubilized in 3× SDS‐sample buffer and separated on 10% SDS polyacrylamide gels. PVDF Membranes (Millipore, Billerica, MA, USA) were incubated with antibodies against DNMT1, SP1, PDPK1 and GAPDH (1:1000). The membranes were washed and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling). The membranes were washed again and transferred to freshly made ECL solution (Millipore) and observed, recorded the signals using the Gel Imagine System (Bio‐Rad, Hercules, CA, USA).
5
Western Blot Analysis of EMT Markers
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Hela and c33a cells were harvested, washed and lysed with radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China). Protein concentration was determined by the Thermo bicinchoninic acid (BCA) protein assay Kit. Equal amounts of proteins from whole cell lysates were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels and then the proteins were transferred onto polyvinylidene fluoride (PVDF) membranes and incubated for immunoblotting with the relevant primary antibodies against Snail, Vimentin and E-cadherin (1:1000 dilutions) at 4°C overnight. The membranes were washed and incubated with a secondary antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling, Beverly, MA, USA). The membranes were washed again and transferred to freshly made enhanced chemiluminescence (ECL) solution (Immobilon Western; Millipore, Billerica, MA, USA), followed by observing signals using the Gel Imagine System (Bio-Rad, Hercules, CA, USA) and documenting the results. Image J software (National Institutes of Health, Bethesda, MD, USA) was applied to quantify and compare the intensity of single band between the control and proteins of interest.
6
Western Blot Analysis of Key Signaling Proteins
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The detailed method was based on previous report [19 (link)]. After measuring the protein concentrations using the Bio-Rad protein method, whole cell lysates containing same amount of protein were solubilized in 4× SDS-sample buffer and separated on 10% SDS polyacrylamide gels. Membranes (Millipore, Billerica, MA, USA) were incubated with antibodies against ERK1/2, AMPKa, pERK1/2, p-AMPKα, FOXO3a and RUNX3 (1:1000). The membranes were washed and incubated with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase (Cell Signaling, Beverly, MA, USA). The membranes were washed again and transferred to freshly made ECL solution (Immobilon Western; Millipore, Billerica, MA, USA) and observed, recorded the signals using the Gel Imagine System (Bio-Rad, Hercules, CA, USA) or exposed to X-ray film.
7
Western Blot Protein Analysis
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Equal amounts of protein from whole cell lysates were dissolved in SDS‐sample buffers and separated on 10% SDS polyacrylamide gels. Membranes (Millipore) were incubated with antibodies against PDPK1 for 2 hours, washed and incubated with secondary rabbit IgG antibody conjugated to horseradish peroxidase (Cell Signaling Technology). The membranes were washed again and transferred to a freshly prepared enhanced chemiluminescence solution (Immobilon Western; Millipore). Protein bands were observed using the Gel Imagine System (Bio‐Rad). ImageJ software (National Institutes of Health) was used to quantify and compare the intensity of single band between the control and proteins of interest.
8
Protein Extraction and Western Blot Analysis
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The clinical specimens were ground with liquid nitrogen. Then, RIPA lysis buffer was introduced to extract the proteins from clinical specimens and cells. The protein concentration was quantified using a BCA detection kit (Junxin Biotech). Protein extract (30 μg) was separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred onto a nitrocellulose membrane (Millipore, Billerica, USA). Membranes were blocked with 5% skim milk, incubated with primary antibodies and horseradish peroxidase (HRP)-labeled secondary antibody successively. The protein bands were visualized using an ECL reagent (Junxin Biotech) on Gel Imagine System (Bio-Rad, Hercules, USA). Antibodies used in this study are shown as follows: anti-DNMT1 (Abcam, Cambridge, UK; ab87656), anti-SOX5 (Millipore; 07e131) and anti-p21 (Abcam; ab109520). The secondary antibody was purchased from Service Biotechnology Co., Ltd (Wuhan, China). The dilution ratio of antibodies was 1:1000.
9
Quantification of Apoptosis Markers by Western Blot
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The determination of protein levels of Bcl-2 and cleaved caspase 3 were done by western blot. Total protein samples were purified from cell lysate and tumor tissues by RIPA lysis buffer (Beyotime, China). Equal amounts of protein samples were separated on SDS-polyacrylamide gels and transferred to the membrane (Millipore, USA). After blocked with 5% skimmed milk for 1 hour at room temperature, the membrane was incubated with primary antibodies to Bcl-2 (1:1000; Abcam, UK) and cleaved caspase 3 (1 µg/ml; Abcam) for 12 h at 4 °C. After washed three times, the membranes were incubated with the Goat Anti-Rabbit IgG H&L (HRP) (1:2000; Abcam) for 1 hour at room temperature. The blots were observed using the gel imagine system (Bio-Rad, CA, USA). GAPDH (1:10000; Abcam) was used as an internal control.
10
Western Blot Analysis of Apoptosis Markers
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The determination of protein levels of Bcl-2 and cleaved caspase 3 were done by western blot. Total protein samples were puri ed from cell lysate and tumor tissues by RIPA lysis buffer (Beyotime, China). Equal amounts of protein samples were separated on SDS-polyacrylamide gels and transferred to the membrane (Millipore, USA). After blocked with 5% skimmed milk for 1 hour at room temperature, the membrane was incubated with primary antibodies to Bcl-2 (1:1000; Abcam, UK) and cleaved caspase 3 (1 µg/ml; Abcam) for 12 h at 4 °C. After washed three times, the membranes were incubated with the Goat Anti-Rabbit IgG H&L (HRP) (1:2000; Abcam) for 1 hour at room temperature. The blots were observed using the gel imagine system (Bio-Rad, CA, USA). GAPDH (1:10000; Abcam) was used as an internal control.
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