The tissues were homogenized in cold T-Per lysis buffer (Pierce Biotechnology, Inc. USA). Approximately 30μg/lane of protein samples were separated by 10–15% SDS-PAGE gel and subsequently transferred to nitrocellulose membranes. After blockage in 5% skim milk powder in 0.1% Tris-buffered saline/Tween 20 (TBST) for 2 h and incubation with antibodies against NLPR3 (D4D8T, Cell Signaling Technology), caspase-1 (sc-392736, Santa Cruz Biotechnology), GSDMD (YT7991, Immunoway Biotechnology), PSMA7 (15219-1-AP, Proteintech Group) overnight at 4°C at a dilution of 1:500 or 1:1000. Then the membranes were incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Immunoreactive proteins were visualized using an enhanced chemiluminescence western blotting detection system (Santa Cruz Biotechnology). The chemiluminescence signal from the membranes was quantified by the GeneGenome HR scanner using GeneTools software (SynGene). To control sampling errors, the ratio of band intensity to β-actin was obtained to quantify the relative protein expression level.
Enhanced chemiluminescence western blotting detection system
Manufactured by Santa Cruz Biotechnology
The Enhanced chemiluminescence western blotting detection system is a laboratory equipment used to detect and analyze proteins in a sample through a western blotting technique. It utilizes chemiluminescence for protein detection and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Genegnome hr scanner, by Syngene (4 mentions)
Sirt1, by Santa Cruz Biotechnology (3 mentions)
Genegenome hr scanner, by Syngene (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Beyotime (2 mentions)
T per lysis buffer, by Thermo Fisher Scientific (1 mentions)
Gsdmd, by Immunoway (1 mentions)
Sc 392736, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg hrp, by Santa Cruz Biotechnology (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
α sma, by Abcam (1 mentions)
Tgf β, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Pgc 1α, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Beyotime (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
8 protocols using enhanced chemiluminescence western blotting detection system
1
Western Blot Analysis of Inflammatory Proteins
2
Quantitative Analysis of Osteoblast Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteoblast proteins were extracted using RIPA buffer including 1% protease inhibitor cocktail (Thermo Fisher Scientific, Inc.) and BCA assay was used to calculate the protein concentration. Then 30 µg proteins per lane was subjected to SDS-PAGE (10% gels) to separate, prior to their transfer onto PVDF membranes. After blocking with nonfat dry milk dissolved in TBS containing 0.05% Tween-20 for 1-2 h at room temperature, the membranes were incubated with antibodies against sirt1 (Santa Cruz Biotechnology, Inc.; cat. no. sc-74665); p53 (ProteinTech Group, Inc.; cat. no. 10442-1-AP) and p21 (ProteinTech Group, Inc.; cat. no. 28248-1-AP) and GAPDH (Santa Cruz Biotechnology, Inc.; cat. no. sc-137179) overnight at 4˚C at a dilution of 1:1,000. Subsequently, the membranes were incubated with a secondary horseradish peroxidase-conjugated antibody for 1-2 h at room temperature at a dilution of 1:2,000, including goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc.; cat. no. sc-2004) and goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc.; cat. no. sc-2005). An enhanced chemiluminescence western blotting detection system (Santa Cruz Biotechnology, Inc.) and a GeneGnome HR scanner (Syngene) were used to visualize the immunoreactive proteins and chemiluminescent signal from the membranes. The expression levels of the proteins of interest were normalized to GAPDH.
3
Protein Expression Analysis of Lung Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples of lung tissues or MLVECs were collected and processed according to the standard protocols. Briefly, 30 μg of protein was separated by 10% SDS-PAGE and subsequently transferred to nitrocellulose membranes (Millipore Corp, Bedford, MA). After blocking, immunoblots were incubated with primary antibody against collagen I (Abcam), E-cadherin (Santa Cruz), α-SMA (Abcam), TGF-β (Cell Signaling, Beverly, MA) or β-actin (Sigma-Aldrich) at 4°C overnight, followed by incubation with a secondary HRP-conjugated IgG (Santa Cruz) for 1 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Santa Cruz).
4
Western Blot Analysis of Osteoblast Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Osteoblastic MC3T3‐E1 cells were lysed using cold RIPA (Beyotime) containing 1% Protease Inhibitor Cocktail (Thermo Fisher) to obtain total protein. A 10% SDS‐PAGE was performed to separate the proteins, which were transferred to PVDF membranes. After blocking with 5% skimmed milk and 0.1% TBST for 2 H, the membranes were incubated with Sirt1 (Santa Cruz), PGC1α (Abcam), or β‐actin (Santa Cruz) antibodies in antibody dilution buffer (Beyome) at 4 °C overnight. The membranes were incubated with a secondary horseradish peroxidase‐conjugated antibody for 1–2 H at room temperature. The enhanced chemiluminescence Western blotting detection system (Santa Cruz) and a GeneGnome HR scanner (SynGene) were used to visualize the immunoreactive proteins and chemiluminescent signal from the membranes, respectively.
5
Bmp2 Expression Analysis in DRG Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The expression of Bmp2 from sham and BCP mice was measured by western blot analysis. Rats were rapidly sacrificed by cervical dislocation. L3-L5 DRGs were quickly harvested and lysed; lysates were rotated for 2 h at 4°C, followed by centrifugation at 12,000 r/min for 15 min at 4°C. Supernatants were collected, and total protein concentration was titrated using a bicinchoninic acid kit. Equivalent amounts of protein (20 μg) were fractionated on 10% polyacrylamide gels. Proteins were transferred to nitrocellulose membranes (Millipore) at 100 mA for 90 min. Membranes were blocked with 5% nonfat milk in Tris-buffered saline (TBS) (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) for 1 h at room temperature and incubated overnight at 4°C with primary antibody (anti-Bmp2 at 1:1000, Abcam) in TBS containing 1% milk. Following three washes with TBST, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:500, Abcam) in TBS containing 1% milk for 2 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence western blotting detection system (Santa Cruz). Membranes were subsequently stripped and reprobed for anti-β-actin antibody (1: 1000, Sigma). The chemiluminescent signal from the membranes was quantified by a GeneGenome HR scanner using GeneTools software (SynGene). Bmp2 protein expression was normalized to β-actin.
6
Western Blot Analysis of NKRF Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The proteins of the H9c2 cardiomyocytes were lysed with cold RIPA lysis buffer (Beyotime). The protein load was 30 µg/lane in 10% SDS-PAGE, and it was subsequently transferred to nitrocellulose membranes. The bolts were blocked with 5% skim milk powder in 0.1%tris-buffered saline/Tween20 for 2 h and incubated with antibodies (Santa Cruz, Cat. No. sc-365568) against NKRF overnight at 4°C at a dilution of 1:1,000. Then, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. The immunoreactive proteins were visualized using the enhanced chemiluminescence western blotting detection system (Santa Cruz). The chemiluminescent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene). To control sampling errors, the ratio of band intensities to β-actin was obtained to quantify the relative protein expression level.
7
Western Blot Analysis of Adrenal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mouse adrenal glands were lysed with cold RIPA lysis buffer (Beyotime). Protein load was 30 μg/lane in 10% SDS-PAGE subsequently transferred to nitrocellulose membranes. The bolts were blocked with 5% skim milk powder in 0.1% Tris-buffered saline/ Tween 20 (TBST) for 2h and incubated with antibodies against iNOS (Santa Cruz), SIRT1 (Santa Cruz) or ACTH receptor (Santa Cruz) overnight at 4˚C at a dilution of 1:1,000. Then, the membrane was incubated with a secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. Immunoreactive proteins were visualized using the enhanced chemiluminescence western blotting detection system (Santa Cruz). The chemiluminiscent signal from the membranes was quantified by a GeneGnome HR scanner using GeneTools software (SynGene). To control sampling errors, the ratio of band intensities to the β-actin was obtained to quantify the relative protein expression level.
8
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chorionic tissues or cells were homogenized in radioimmunoprecipitation assay lysis buffer containing phosphatase inhibitor cocktail (Roche, Indianapolis, IN). Proteins (50 mg) were denatured and separated by 12% SDS-PAGE and subsequently transferred to nitrocellulose membranes by electroblotting. The membranes were incubated in blocking buffer, and then with specific antibodies against CBS (Abcam, Cambridge, UK), CSE (Abcam), and PGDH (Cayman Chemical, Ann Arbor, MI) at 4 C overnight; finally, they were incubated with a secondary horseradish peroxidaseeconjugated IgG (1:1000). Immunoreactive proteins were visualized using the enhanced chemiluminescence Western blotting detection system (Santa Cruz Biotechnology). The intensities of light-emitting bands were detected and quantified using Sygene Bio Image system (Synoptics Ltd, Cambridge, UK). To control sampling errors, the ratio of band intensities/b-actin was obtained to quantify the relative protein expression level.
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!