Sub-confluent monolayers of cells were lysed and supernatants were recovered by centrifugation. Protein concentrations were measured using the BCA protein assay kit (Thermo Scientific, Waltham, MA) and equal amounts of total protein were resolved using SDS-PAGE gels. Resolved proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) and blocked with 5% milk in TBS/Tween-20 (TBST). Membranes were incubated with the following primary antibodies: DDR1 (1:1,000 dilution, #5583, Cell Signaling Technology, Danvers, MA), phosphorylated-DDR1 (1:1,000 dilution, #11994, Cell Signaling Technology), phosphorylated-PYK2 (1:1,000 dilution, #3291, Cell Signaling Technology), E-cadherin (1:2,000, 610181, BD Biosciences), and beta-actin (1:5,000 dilution, sc-47778, Santa Cruz Biotechnology), followed by the corresponding HRP-conjugated secondary antibodies (Jackson ImmunoResearch Labs, West Grove, PA). Protein bands were detected using the enhanced chemiluminescence reagent kit (Thermo Scientific) on autoradiographic films.
Enhanced chemiluminescence reagent kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Enhanced chemiluminescence reagent kit is a laboratory product that provides a solution for the detection and quantification of proteins using chemiluminescence technology. The kit contains the necessary reagents for the chemiluminescent reaction, which can be used to visualize and analyze protein samples.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Santa Cruz Biotechnology (2 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
E cadherin, by BD (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Phosphorylated pyk2, by Cell Signaling Technology (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Caspase 1, by ABclonal (1 mentions)
Nlrp3, by Abcam (1 mentions)
Il 1β, by ABclonal (1 mentions)
Hsp90, by BD (1 mentions)
Ibright fl100 imaging system, by Thermo Fisher Scientific (1 mentions)
Phosphorylated smad2 3, by Cell Signaling Technology (1 mentions)
Total smad2 3, by Cell Signaling Technology (1 mentions)
Hrp conjugated goat anti rabbit secondary antibody, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
7 protocols using enhanced chemiluminescence reagent kit
1
Western Blot Analysis of DDR1, PYK2, and E-cadherin
2
Quantifying SKA1/2/3 Protein Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total proteins were extracted and quantified from cultured cells utilizing lysis buffer and the Pierce BCA protein assay kit (Thermo, USA), respectively. The protein extracts (20 mg) were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked 5% nonfat milk buffer, and then probed with primary antibodies against SKA1 (dilution: 1:1000, ab91550), SKA2(dilution: 1:1000, ab75345), and SKA3(dilution: 1:1000, ab186003) which were all purchased from Bioss Company. After incubation with secondary anti-rabbit antibodies or anti-mouse, protein bands were visualized using an enhanced chemiluminescence reagent kit (Thermo, USA). The relative expressions of SKA1/2/3 were detected employing a chemiluminescence detection system in line with the manufacturer’s instructions.
3
Lung Protein Expression Analysis in Rats
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The total proteins of fresh lung tissues in rats were extracted by radio immunoprecipitation assay lysis fluid (Beyotime, No. P0013 B). Then the samples were centrifuged at 12,000 rpm at 4°C for 10 min. After the separation of SDS-polyacrylamide gel electrophoresis buffer, they were transferred to a polyvinylidene fluoride (PVDF) membrane. After the transfer solution on the membrane was washed away, western blocking solution was added, and the mixture was slowly shaken on a shaking table and blocked at room temperature for 2 hours. The membrane was incubated with the following primary antibodies at 4°C overnight: ACE2 (1 : 500), MasR (1 : 500), NF-κB (1 : 1000), and β-actin (1 : 500), which were then incubated with horseradish peroxidase-conjugated secondary antibodies (Goat anti-rabbit IgG, 1 : 20000 dilution) for 1.5 h, followed by the addition of washing buffer (PBST) for 10 minutes each and 3 washings in total. Corresponding protein expressions were measured using an enhanced chemiluminescence reagent kit (Thermo, No. 34094). The semiquantitative analyses of immunoblots were performed through Image J.
4
Quantifying NLRP3, Caspase-1, and IL-1β in HCEC Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the lysed samples of HCEC cells in RIPA buffer containing protease inhibitors (Beyotime, Jiangsu, China). Western blotting was performed as our previously description42 (link)–44 (link), with the following antibodies including: NLRP3 (1:1,000; Cat#ab263899, Abcam), caspase-1 (1:1,000; Cat#A0964 Abclonal, Wuhan, China), IL-1β (1:1,000; Cat#A11369, Abclonal) and anti-rabbit IgG HRP-linked Antibody (1:3,000; Cat#7074, Cell Signaling Technology, MA, USA). The specific bands were visualized by an enhanced chemiluminescence reagent kit (Cat#34094, Thermofisher). The western blot signals are 16-bit images captured by a Luminescent Imaging Workstation (Tanon, Shanghai, China).
5
Western Blot Analysis of Cell and Tissue Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A7r5 cells and mouse aortic tissue were lysed with RIPA buffer containing protease and phosphatase inhibitors under reducing condition. Protein content was measured with a Bradford assay. Protein samples and molecular weight standards were separated by 1‐dimensional gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes, which were blocked for 2 hours in 5% nonfat dry milk dissolved in Tris‐buffered saline (pH 7.4) with 1% TWEEN 20. Membranes were then incubated in 5% nonfat dry milk for 2 hours at room temperature with antibodies to translin and trax, which were generated in our lab,21 phosphorylated‐SMAD2/3 (catalog no. 8828) and total‐SMAD2/3 (catalog no. 8685) from Cell Signaling Technologies (Danvers, MA); Hsp90 (catalog no. 610419, BD Transduction Laboratories, Franklin Lakes, NJ). Membranes were incubated with secondary antibody with the appropriate horseradish peroxidase–conjugated IgG in Tris‐buffered saline with 1% TWEEN 20 with 5% nonfat dry milk for 1 hour at room temperature. Immunoreactive proteins were visualized using an enhanced chemiluminescence reagent kit (catalog no. 34577, ThermoFisher Scientific). The signals emitted for chemiluminescence were detected with the iBright FL100 Imaging system (ThermoFisher Scientific), and band density was analyzed with ImageJ.
6
Protein extraction and Western blot analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We use RIPA buffer (Thermo Scientific, USA) to extract protein from cells according to the manufacture's protocols. After determination of protein concentration using BCA assay (Thermo Scientific, USA), the supernatants were mixed with loading buffer. In brief, the mixture containing 20 μg protein were used for separation on 10% SDS-PAGE, and then electro-transferred onto NC membranes (Millipore, USA). The membranes were then blocked in 5% non-fat milk for 1 h at room temperature and then incubated with primary antibodies (β-actin (Santa Cruz): 1: 500; SOD2 (Cell Signaling Technology): 1: 1000; SIRT3 (Cell Signaling Technology): 1: 1000; PGC-1ɑ (Abcam): 1:1000) overnight at 4 °C. After that, the membranes were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:5000. Thermo Scientific, USA) for 30 min at 37 °C. The bands were detected using an enhanced chemiluminescence reagent kit (Thermo Scientific, USA).
7
Western Blot Analysis of c-Kras Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein for western blot analysis was precipitated according to the standard protocol (15 (link)). Equal quantities of protein samples were subjected to SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membrane was soaked in Tris-buffered saline and Tween-20 (TBST) buffer containing 5% low-fat milk for 60 min with gentle agitation. The membrane was then incubated with monclonal rabbit anti-human c-Kras (1:1,000) and mouse anti-human GAPDH (1:1,000) antibodies (Cell Signaling Technologies, Inc., Danvers, MA, USA) overnight followed by washing with TBST buffer and a further incubation with monoclonal rabbit anti-mouse and mouse anti-rabbit secondary antibodies (1;10,000; Cell Signaling Technologies, Inc.). Finally, an enhanced chemiluminescence reagent kit (Thermo Scientific, Waltham, MA, USA) was used to detect of the protein bands, which were quantified by densitometry (Image Lab™ analysis software; Bio-Rad, Hercules, CA, USA), normalized to GAPDH and expressed as the fold of the control. The primary antibodies used were rabbit anti-c-Kras (1:1,000) and mouse anti-GAPDH (1:1,000). The secondary antibodies were rabbit anti-mouse (1:10,000) and mouse anti-rabbit (1:10,000). All of the antibodies were purchased from Cell Signaling Technology, Inc.
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!