After plasma treatment for 48h, cell protein was extracted using RIPA lysis buffer (Sigma), and the concentration was determined by BCA protein assay kit (Sigma). Protein samples were denatured with 5×protein loading buffer at 95°C for 5min and separated in 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). Subsequently, proteins were transferred to 0.2 μm of polyvinylidene difluoride membranes (Millpore, Billerica, MA, USA) at 70 V for 2.5 h. After blocking with 5% non-fat milk for 2 h at room temperature, the membrane was incubated with primary antibody and then washed with TBST three times. The horseradish peroxidase (HRP)–conjugated secondary antibodies, goat anti-rabbit IgG and anti-mouse IgG (1:2000) (Abgent, San Diego, CA, USA), were added and incubated for 1h on a horizontal orbital shaker. Images were visualized with ECL chemiluminescent substrate (Millipore, Bedford, MA, USA) using a ChemiDoc-It 510 system (UVP, Upland, CA, USA) and protein bands were analyzed by densitometric analysis using ImageJ software. Primary antibody against human included caspase-3 (1:500), caspase-8 (1:500), caspase-9 (1:500), phospho-p53 (1:200) (Cell Signaling Technology, Danvers, MA, USA), CD95 (1:200) (Santa Cruz, CA, USA) and β-actin (1:1000) (Immuno Way, Newark, DE, USA).
Ecl chemiluminescent substrate
Manufactured by Merck Group
Sourced in United States
ECL chemiluminescent substrate is a laboratory reagent used to detect and quantify proteins in Western blot analysis. It emits light when exposed to an enzyme, allowing for the visualization and measurement of specific proteins.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Microplate reader, by Thermo Fisher Scientific (2 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Phospho p53, by Cell Signaling Technology (1 mentions)
Bca protein assay kit, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Caspase 8, by Cell Signaling Technology (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Anti mouse igg, by Abcepta (1 mentions)
Chemidoc it 510 system, by Analytik Jena (1 mentions)
Caspase 9, by Cell Signaling Technology (1 mentions)
β actin, by Immunoway (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Western and ip lysate, by Beyotime (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Beyotime (1 mentions)
Hrp conjugated goat anti mouse igg secondary antibody, by Beyotime (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Transwell chamber, by Corning (1 mentions)
15 protocols using ecl chemiluminescent substrate
1
Protein Expression Analysis in Cells
2
Western Blot Analysis of Atrial Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total protein of left atrial tissue was extracted after tissue lysis with Western and IP lysate (Beyotime). After the protein was quantified using the bovine serum albumin kit (Beyotime), 20 μg of protein was boiled with loading buffer and subjected to 12% polyacrylamide gel electrophoresis. The protein was then transferred to the nitrocellulose membrane (Millipore, UK) and blocked with 5% skimmed milk at 4°C overnight. Then, appropriately diluted primary antibodies (HCN2 [Bioss]: 1:500; HCN4 [Bioss]: 1: 250; glyceraldehyde 3‐phosphate dehydrogenase [GAPDH] [Santa Cruz]: 1: 500) were added and incubated at room temperature for 2 h. After washing the membrane with 1 × tris buffered saline with tween (TBST) for 10 min three times, the corresponding secondary antibody HRP Conjugated Rabbit Anti‐Goat IgG (H + L) [A21030; Abbkine; 1:1000] was added and incubated at room temperature for 1 h. The membrane was then again subjected to washing with 1 × TBST for 10 min three times. Efficient chemiluminescence (ECL) chemiluminescent substrate (Millipore) was prepared, and the ChemiDoc‐It HR 410 imaging system (Upland, CA) was used to analyze the results.
3
Nrf-2 and HO-1 Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were stimulated as previously indicated for 24 h and intracellular proteins were extracted employing Tris–HCl buffer pH 7.5 with protease inhibitor cocktail and phenylmethylsulfonyl fluoride (all from Sigma-Aldrich). Thereafter, proteins were separated by SDS-PAGE electrophoresis as previously described [46 ]. Once separated, proteins were transferred to membranes and then incubated with the following antibodies: anti-human rabbit Nrf-2 (1:1000) (SantaCruz Biotechnology) and HO-1 (1:1000) (Enzo Life Sciences, New York, USA) overnight at 4 °C and anti-human mouse tubulin (1:1000) (Sigma-Aldrich) 1 h at room temperature. Anti-rabbit or mouse secondary antibody (1:1000, Dako, Germany) and ECL chemiluminescent substrate (Millipore, USA) were used to detect antigen–antibody binding. Protein bands were quantified by densitometry with the ImageQ image processing software (http://imagej.nih.gov/ ). All protein band intensities were normalized to the tubulin band intensity for the same sample.
4
Evaluating PDCL3 Expression and Cell Proliferation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Microplate Reader (Thermo Fisher, USA); ChemiDoc Imaging System (BIORAD, USA); Reverse Transcriptase (Wuhan Miaoling Biotechnology Co., Ltd.); RIPA Lysis Buffer, BCA Protein Assay Kit (Shanghai Epizyme Biotechnology Co., Ltd.); ECL Chemiluminescent Substrate (Milipore, USA); Cell Counting Kit8 Cell Proliferation Assay Kit (Suzhou UElandy Biotechnology Co., Ltd.); Crystal Violet Staining Solution (Shanghai Beyotime Biotechnology Co., Ltd.); Transwell Chambers (Corning, USA); Rabbit Anti-Human PDCL3 Monoclonal Antibody (Proteintech, USA); Mouse Anti-Human GAPDH Polyclonal Antibody (Proteintech, USA); HRP-conjugated Goat Anti-Rabbit IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.); HRP-conjugated Goat Anti-Mouse IgG Secondary Antibody (Shanghai Beyotime Biotechnology Co., Ltd.)
5
Western Blotting Analysis of SOX2 Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blotting analysis, 106 cells were lysed in Laemmli loading buffer (Sigma Aldrich). The cell extract was then sonicated and heated for 5 min at 95° C. Cell lysates were separated by SDS-PAGE using 9% Bis-Tris polyacrylamide gels. Proteins were transferred to Amersham Protran nitrocellulose membranes (0.20 μm-pore size; GE Healthcare, Little Chalfont, UK). Immunoblots were probed overnight at 4 °C in TBS-Tween (100 mmol·L−1 Tris–HCl, 150 mmol·L−1 NaCl and 0.1% Tween-20, pH 7.6) with 3% fat milk with one of the antibodies listed in Table S1 . Membranes were washed and incubated for 1 h at 4 °C with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000, GE Healthcare, Little Chalfont, UK). Membranes were washed three times for 5 min per wash with TBS-Tween, and bound antibodies were detected using ECL chemiluminescent substrate (Immobilon, Millipore, Billerica, MA, USA). Results were analyzed with GeneGnome XRQ (SYNGENE Ozyme, Cambridge, UK). Quantification was performed using GeneTools from syngene. SOX2 expression was normalized to GAPDH for each sample, and then the normalized SOX2 expression in the treated samples was expressed relative to DMSO (control) treatment.
6
RNA-Protein Interaction Analysis by RIP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were prepared in lysis buffer containing 50 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1% (v/v) NP‐40, and 0.5 mM EDTA supplemented with 1× complete protease inhibitor cocktail (Roche) and phosphatase inhibitor PhosSTOP (Roche). Equal total protein amounts from clarified lysates were resolved on SDS‐PAGE, and transferred to 0.2 μm PVDF membranes (Millipore) for Western blot analysis. The target proteins were visualized using ECL chemiluminescent substrate (Millipore) or Odyssey Imaging Systems (LI‐COR Biosciences). For RIP experiments, cell lysates were harvested in lysis buffer with 10 U RNase Inhibitor (Thermo Fisher Scientific), then performed as the introduction of Magna RIP Kit (Millipore) by 5 μg of anti‐PKR or 2 μg of anti‐flag antibodies with equal amount of isotype anti‐IgG, and using 20 μl Protein A/G Magnetic beads. Protein‐RNA complexes were treated with Proteinase K at 55°C for 30 min to elute and extract binding RNA. RNA samples were reverse transcribed and analyzed by qPCR. Results were normalized to input RNA extracted from cell lysates for RIP groups and isotype anti‐IgG groups.
7
Protein Expression Profiling of Mitochondrial Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
By homogenization and lysis in RIPA Lysis Buffer (Beyotime), we extracted total proteins from lung tissues, primary ATII cells, and A549 cells. The protein content was quantified using a BCA kit (Beyotime). Western Blot analysis in lung tissues and cells was performed against DRP1, phosphorylated-DRP1 (p-DRP1) (Ser616), MFF, OPA1, MFN2 (1:1000, Cell Signaling Technology, Danvers, MA, USA), PINK1, PARK2, SQSTM1/p62, LC3b, P16, H2AX (1:1000, Abcam Cambridge, MA, USA), Klotho and GAPDH (1:1000, Proteintech, Wuhan, Hubei, China). Bands were developed by ECL chemiluminescent substrate (Millipore, Billerica, MA, USA).
8
SDS-PAGE and Western Blotting for Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The concentration of the proteins was adjusted with sample buffer (0.125 M Tris-HCl; 4% SDS; 20% v/v glycerol; 0.2 M DTT; 0.02% bromophenol blue; pH 6.8). The proteins were loaded into 12% SDS-polyacrylamide gels for electrophoresis, transferred to nitrocellulose membranes, and stained with Ponceau S. The primary antibodies used were: anti-asyn 1:1000 (BD Healthcare, Franklin Lakes, NJ, USA) and anti-FLAG 1:500 (Sigma-Aldrich, St. Louis, MO, USA). HRP-conjugated secondary antibodies (anti-mouse or anti-rabbit) were purchased from Sigma-Aldrich. The development of immunoblots was performed using ECL chemiluminescent substrate (Millipore, Billerica, MA, USA).
9
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the cells using RIPA lysis buffer (Beyotime, China). The protein concentration was quantified using a dual bicinchoninic acid protein assay kit (Beyotime, China). Protein electrophoresis was performed on 6% and 10% (w/v) sodium dodecyl sulfate–polyacrylamide gels (Vazyme, China). The separated proteins were transferred onto polyvinylidene fluoride membranes (Millipore, USA) and blocked with 5% skim milk powder (Millipore) at room temperature for 2 h. Subsequently, the blocked membranes were incubated with specific primary antibodies overnight at 4 ℃, followed by incubation with secondary antibodies at room temperature for 1.5 h. The following antibodies were used: PITPNC1 (1:800, Sigma, USA), FASN (1:1200, Proteintech, China), CD155 (1:1000, CST, USA), Tubulin (1:5000, Proteintech, China), BCL-2 (1:1000, Abcam, USA), Cleaved Caspase-3 (1:1000, Abcam, USA), Bax (1:1000, Immunoway, USA), and goat anti-rabbit and goat anti-mouse secondary antibody (1:10000, Fude, China). After washing three times for 5 min each with TBST, the proteins were visualized using an ECL chemiluminescent substrate (Millipore, USA) on an Alliance imaging system (UVITEC, UK), and the densities were quantified using ImageJ software. Tubulin served as the internal reference protein.
10
Kidney Protein Quantification and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
By centrifuging samples
at 12,000g for 8 min at 4 °C, protein was collected
from kidneys, and quantification was performed using the Bradford
method Bio-Rad Protein Assay (Hercules, CA) following the manufacturer’s
instructions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was then performed on protein extracts (50 mg protein/sample),
and poly(vinylidene fluoride) (PVDF) membranes were applied (Millipore).
Primary antibodies were applied to the blots (1:1000) except for anti-AMPK
(1:2000), anti-LC3-II (1:500), and anti-TGF-β (1:500), followed
by secondary antibodies (1:5000). To detect specific proteins, ECL
chemiluminescent substrate (Millipore, MA) was used according to the
manufacturer’s instructions. Quantification was done with Image
Quant version 5.1.
at 12,000g for 8 min at 4 °C, protein was collected
from kidneys, and quantification was performed using the Bradford
method Bio-Rad Protein Assay (Hercules, CA) following the manufacturer’s
instructions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) was then performed on protein extracts (50 mg protein/sample),
and poly(vinylidene fluoride) (PVDF) membranes were applied (Millipore).
Primary antibodies were applied to the blots (1:1000) except for anti-AMPK
(1:2000), anti-LC3-II (1:500), and anti-TGF-β (1:500), followed
by secondary antibodies (1:5000). To detect specific proteins, ECL
chemiluminescent substrate (Millipore, MA) was used according to the
manufacturer’s instructions. Quantification was done with Image
Quant version 5.1.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!