Cells and tissues were subjected to RIPA Lysis Buffer (Beyotime) treatment to extract their total proteins. The protein concentrations were then estimated with the aid of the BCA Protein Assay Kit (Beyotime). Afterward, the 25 μg of proteins per lane were electrophoresed through a 10% SDS-polyacrylamide gel. The separated proteins were moved onto nitrocellulose membranes and then treated with 10% skimmed milk solution for 2 h at room temperature. Next, the membranes were maintained at a temperature of 4°C overnight, together with GAPDH (1:2,500 dilution; #ab9485; Abcam) and rabbit anti-human RPN1 (1:2,000 dilution; #ab197888; Abcam) antibodies. On the following day, goat anti-rabbit IgG-HRP (1:5,000 dilution: #ab6721; Abcam) was added onto the membranes before they were incubated for 2 h at room temperature. GAPDH was the internal reference. The immunoreactive bands were imaged with an ECL chemiluminescence solution (Beyotime) and then analyzed in Image Lab Software.
Ecl chemiluminescence solution
Manufactured by Beyotime
Sourced in China
ECL chemiluminescence solution is a reagent used in western blotting analysis to detect and quantify proteins. It generates a chemiluminescent signal upon reaction with the enzyme horseradish peroxidase, which is commonly used to label target proteins. The intensity of the signal is proportional to the amount of the target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (4 mentions)
Ab9485, by Abcam (2 mentions)
Ab6721, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Mouse anti actin, by Transgene (1 mentions)
Rabbit anti arg1, by ABclonal (1 mentions)
Protease inhibitor, by Transgene (1 mentions)
Sds page gel, by Solarbio (1 mentions)
Primary and secondary antibodies, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ab208777, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Igf 1, by Abcam (1 mentions)
γ globulin, by Abcam (1 mentions)
Stem cell factor (scf), by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Abcam (1 mentions)
Cxcr4, by Abcam (1 mentions)
Cxcl12, by Abcam (1 mentions)
Ab182858, by Abcam (1 mentions)
Ab32503, by Abcam (1 mentions)
8 protocols using ecl chemiluminescence solution
1
Protein Quantification and Western Blotting Protocol
2
Protein Expression Analysis in Cell Cultures
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Cells in one well of a six-well plate were prepared for each group. Cells were lysed using RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor (TransGen Biotech, Beijing, China). After agarose gel electrophoresis, fibronectin, ZO-1, collagen 1A1, vimentin, ARG1, and β-actin were detected using the primary antibodies rabbit anti-fibronectin (1:500; ABclonal, Wuhan, China), rabbit anti-ZO-1 (1:500; ABclonal), rabbit anti-ARG1 (1:500; ABclonal), mouse anti-vimentin (1:500; Beyotime), rabbit anti-collagen 1A1 (1:500; Huabio, Hangzhou, China), and mouse anti-actin (1:1000; TransGen Biotech, Beijing, China). The PVDF membrane was then incubated with secondary antibodies for 1 h at room temperature. After the membrane was washed, the protein bands were detected using ECL chemiluminescence solution (Beyotime). The grey value for each protein was normalized to that of β-actin and averaged. Each experiment was independently repeated three times.
3
Protein Extraction and Western Blot Analysis
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Protein extraction
was conducted using a RIPA lysis buffer (Beyotime, China) along with
the addition of both phosphatase and protease inhibitors. Following
protein extraction, precise protein concentration measurements were
carried out by utilizing a BCA protein quantification kit. Subsequently,
protein samples were combined with a 10% SDS-PAGE gel (Solarbio, China)
to effect protein separation through gel electrophoresis. Upon completion
of the separation process, proteins were transferred onto poly(vinylidene
fluoride) (PVDF) membranes to facilitate subsequent immunodetection.
To mitigate nonspecific binding, PVDF membranes were subjected to
a blocking procedure involving 5% skim milk. During the immunodetection
phase, specific antibodies were incubated with the target protein
on the PVDF membrane, a process carried out overnight at 4 °C.
Afterward, suitable secondary antibodies were used to enhance the
signal following the primary antibody reaction. Visualization of protein
bands was achieved through the utilization of an ECL chemiluminescence
solution (Beyotime), enabling reliable visualization of protein bands.
Ultimately, the quantitative analysis of protein bands was performed
using ImageJ software.
was conducted using a RIPA lysis buffer (Beyotime, China) along with
the addition of both phosphatase and protease inhibitors. Following
protein extraction, precise protein concentration measurements were
carried out by utilizing a BCA protein quantification kit. Subsequently,
protein samples were combined with a 10% SDS-PAGE gel (Solarbio, China)
to effect protein separation through gel electrophoresis. Upon completion
of the separation process, proteins were transferred onto poly(vinylidene
fluoride) (PVDF) membranes to facilitate subsequent immunodetection.
To mitigate nonspecific binding, PVDF membranes were subjected to
a blocking procedure involving 5% skim milk. During the immunodetection
phase, specific antibodies were incubated with the target protein
on the PVDF membrane, a process carried out overnight at 4 °C.
Afterward, suitable secondary antibodies were used to enhance the
signal following the primary antibody reaction. Visualization of protein
bands was achieved through the utilization of an ECL chemiluminescence
solution (Beyotime), enabling reliable visualization of protein bands.
Ultimately, the quantitative analysis of protein bands was performed
using ImageJ software.
4
Protein Expression Analysis via Western Blot
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The cells were cultured in 6-well plates and treated with 200 µM EFL1 or EFL2 for 72 h. Cells were lysed by RIPA and the protein concentrations were quantified by BCA method. Equal amounts of 20 μg proteins were loaded for separation by SDS-page electrophoresis, and then electrophoretically transferred to polyvinylidene difluoride membranes. After blocking in 5% skim milk and successively incubation with primary and secondary antibodies (Proteintech and Abclonal, Wuhan, China), the membranes were exposed to ECL chemiluminescence solution (Beyotime, Shanghai, China) for imaging.
5
Western Blot Analysis of GRN Protein
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Cells were lysed by RIPA lysis buffer (Beyotime, Shanghai, China), and total cell proteins were extracted after the centrifugation and the concentration of the protein in the supernatant was determined by BCA method. After the loading buffer was added, the supernatant was immediately heated in a water bath at 100°C for 10 min to denature the protein. Subsequently, the protein was separated via SDS-PAGE and subsequently transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA), which was then blocked with 5% skimmed milk for 1 h at room temperature and then incubated overnight at 4°C with the primary anti-GRN antibody (ab208777, 1: 500, Abcam, Cambridge, UK) and anti-GAPDH antibody (ab9485, 1: 2000, Abcam, Cambridge, UK). Furthermore, rinsed with TBST, the membrane was incubated with Goat Anti-Rabbit IgG H&L (ab205718, 1:1000, Abcam, Cambridge, UK) at ambient temperature for 1 h, and washed by TBST. Eventually, ECL chemiluminescence solution (Beyotime, Shanghai, China) was added on the PVDF membrane, and the protein bands were developed, with GAPDH as the internal reference. Ultimately, the gray values were calculated by ImageJ (NIH, Bethesda, Maryland, USA).
6
Western Blot Protein Analysis
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Cells were collected in the logarithmic growth phase, RIPA lysis buffer was used to extract total cell protein, and the protein concentration was determined by using the BCA kit. 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins and transfer to the PVDF membrane, and they are incubated in 5% skimmed milk for 1 hour. Diluted primary antibodies (α-globulin, γ-globulin, CSF2, CXCR4, DKK1, CXCL12, IGF1, and SCF, 1 : 1000, Abcam, Cambridge, MA, USA) were left overnight at 4°C, then horseradish peroxidase- (HRP-) conjugated secondary antibody (1 : 10000, Abcam, Cambridge, MA, USA) was added, and they were incubated for 1 hour. Next, we applied ECL chemiluminescence solution (Beyotime, Shanghai, China) for color development. Images were collected in a gel imaging system.
7
Protein Extraction and Western Blot Assay
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After the cells were processed in different factors, the old culture solution was discarded, and the cells were trypsinized with the lysate of RIPA (containing 1% PMSF) (Beyotime Biotechnology, Shanghai, China) and collected by centrifugation to extract the total protein. After total protein was extracted, a BCA protein concentration kit (Beyotime Biotechnology, Shanghai, China) was used to quantify protein concentration. Twenty microgram total protein in each group was subjected to SDS-PAGE for the separation of proteins. When the electrophoresis was finished, the isolated protein was transferred to the PVDF membranes at 200 mA for 90 min. Then, the membranes were blocked with a 10% skim milk powder solution for 2 h, the AGR2 monoclonal antibody (Abcam, ab76473; 1:1000), HIF-1A monoclonal antibody (Abcam, ab179483; 1:1000), Cleaved Caspase-3 polyclonal antibody (Abcam, ab2302; 1:1000), Bcl2 polyclonal antibody (Abcam, ab182858; 1:1000), Bax monoclonal antibody (Abcam, ab32503; 1:1000) were used for incubation overnight at 4°C. Next, TBST was applied to wash the membranes for three times (15 min each time), and the membranes were incubated with the secondary antibody for 2 h at room temperature and washed three times with TBST, and protein blots were exposed using an ECL chemiluminescence solution (Beyotime Biotechnology, Shanghai, China).
8
Western Blot Protein Analysis
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The total protein was extracted with RIPA lysis and extraction buffer (Thermo Fisher, Waltham, MA, USA), and the protein concentration was detected by bicinchoninic acid kit (Abcam, Cambridge, MA, USA). 10%sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was selected to separate the protein according to the molecular weight of the target protein. After electrophoresis, the protein was transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk powder at room temperature for 90 min, the primary antibody was added at 4 °C overnight (1:1,000) (Abcam, Cambridge, MA, USA). The following day, the secondary antibody linked to horseradish peroxidase (1:1,000) (Abcam, Cambridge, MA, USA) was incubated at room temperature for 1 h, and finally placed in ECL chemiluminescence solution (Beyotime, Shanghai, China) for imaging.
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