The membrane fraction samples (input samples) and the samples obtained after CoIP were subjected to SDS-PAGE using a Tris-glycine gel. The samples were mixed with the loading buffer and 2-mercaptoethanol and heated at 95 °C for 5 min prior to loading. After electrophoretic separation, proteins were electrophoretically transferred onto PVDF membrane. The membrane was blocked with 5% (w/v) skim milk in 20 mM Tris-HCl (pH 7.4), 150 mM NaCl containing 0.1% (v/v) Tween-20 (TBS-T buffer) for 60 min. After washing the membrane with TBS-T, primary antibodies, diluted in TBS-T containing 5% (w/v) skim milk, were added, and the membrane was incubated overnight at 4 °C. Anti-DDDDK-tag mAb HRP-DirecT (MBL), which binds to the FLAG tag, was used to detect PRELP, IGF-I Receptor β rabbit mAb (Cell Signaling Technology) was used to detect IGFI-R, and p75NTR rabbit mAb (Cell Signaling Technology) was used to detect p75NTR. As a loading control for input samples, Na,K-ATPase was detected by Na,K-ATPase α1 rabbit mAb (Cell Signaling Technology). Anti-DDDDK-tag mAb HRP-DirecT was detected by the ECL detection system (Amersham) according to the manufacturer's instruction. The primary antibodies that recognize IGFI-R, p75NTR, and Na,K-ATPase were detected using anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology) using the ECL detection system.
Ecl detection system
Manufactured by Cell Signaling Technology
Sourced in United States
The ECL detection system is a laboratory equipment designed for chemiluminescent detection in Western blotting applications. It functions by detecting and measuring light emission from a chemiluminescent reaction, allowing for the visualization and analysis of protein targets.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (6 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Enhanced ripa buffer, by Beyotime (1 mentions)
Fv1000 confocal microscope system, by Olympus (1 mentions)
Bradford assay, by Beyotime (1 mentions)
Anti β actin polyclonal antibody, by Santa Cruz Biotechnology (1 mentions)
Anti vimentin, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin, by Santa Cruz Biotechnology (1 mentions)
Anti n cadherin, by Santa Cruz Biotechnology (1 mentions)
Quantity one 4, by Bio-Rad (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Pro prep, by iNtRON Biotechnology (1 mentions)
Anti actin, by Cell Signaling Technology (1 mentions)
Anti gfp, by Takara Bio (1 mentions)
Ag019, by Beyotime (1 mentions)
Ab236422, by Abcam (1 mentions)
Ab245309, by Abcam (1 mentions)
Ab112034, by Abcam (1 mentions)
11 protocols using ecl detection system
1
Western Blot Analysis of Protein Complexes
2
Axl Protein Detection in Tumor Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole cell protein lysate was obtained by enhanced RIPA buffer (Beyotime). Samples were sonicated and then centrifuged at 15,000 g for 10 min at 4°C. Protein concentrations were determined by Bradford assay (Beyotime). Equal amounts of protein were fractionated by SDS-PAGE, transferred to a PVDF membrane (Millipore), and analyzed by incubation with primary rabbit anti-mouse/human Axl antibody. Proteins were detected via incubation with HRP-conjugated secondary antibodies and enhanced chemiluminescence (ECL) detection system (Cell Signaling Technology).
For immunofluorescence staining, tumor cells were seeded on collagen-coated glass slides and grown to 50–60% confluence. Cells were then fixed in fresh 3% formaldehyde in PBS followed by permeabilization and blocking in Tris-buffered saline + 0.05% Tween 20 (TBST) containing 5% bovine serum albumin and 0.3% Triton X-100 at room temperature. After that, cells were stained with primary rabbit anti-mouse/human Axl antibody followed by secondary FITC-labeled goat anti-rabbit antibody. After final washes in TBST, cells were mounted in ProLong Gold anti-fade reagent (Life Technologies) and imaged on the Olympus FV1000 confocal microscope system. Acquired images were processed by ImageJ software.
For immunofluorescence staining, tumor cells were seeded on collagen-coated glass slides and grown to 50–60% confluence. Cells were then fixed in fresh 3% formaldehyde in PBS followed by permeabilization and blocking in Tris-buffered saline + 0.05% Tween 20 (TBST) containing 5% bovine serum albumin and 0.3% Triton X-100 at room temperature. After that, cells were stained with primary rabbit anti-mouse/human Axl antibody followed by secondary FITC-labeled goat anti-rabbit antibody. After final washes in TBST, cells were mounted in ProLong Gold anti-fade reagent (Life Technologies) and imaged on the Olympus FV1000 confocal microscope system. Acquired images were processed by ImageJ software.
3
Protein Expression Analysis in FaDu Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from tissues and FaDu cells for protein analysis. The samples were boiled at 100°C for 5 min. An equal amount of protein samples was separated by SDS-PAGE and transferred to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies overnight at 4°C. The antibodies used were as follows: anti-ADAM10 (1:300, polyclonal, rabbit anti-human; BBI Life Sciences Corporation, Shanghai, China), anti-Ki-67 (1:1,000; polyclonal, rabbit anti-human), anti-E-cadherin (1:1,000), anti-N-cadherin (1:1,000, monoclonal, mouse anti-human), anti-vimentin (1:1,000, monoclonal, mouse anti-human), and anti-β-actin polyclonal antibody (1:2,500, monoclonal, mouse anti-human) (all from Santa Cruz Biotechnology, USA). Immunoreactive protein bands were detected using an ECL detection system (Cell Signaling Technology, USA).
4
Protein Expression Analysis of Wound Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the epithelium of the wound tissue using PRO-PREP (Intron Biotechnology, Inc., Seongnam, Korea), according to the manufacturer's protocol and the BCA protein determination method was used. Equal amounts of protein (30 µg per lane) were separated by 12% SDS-PAGE, transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA) and blocked with 10% bovine serum albumin (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at room temperature for 2 h. Membranes were then incubated with antibodies specific to GLUT1 (1:1,000; cat no. NB110-39113; Novus Biologicals Canada ULC, Oakville, ON, Canada), vascular endothelial growth factor (VEGF) (1:500; cat no. MAB293R; R&D Systems, Inc., Minneapolis, MN, USA) and GAPDH (1:2,000; cat no. GTX100118; GeneTex, Inc., Irvine, CA, USA) overnight at 4°C, followed by incubation with anti-rabbit Immunoglobulin G horseradish peroxidase-conjugated antibodies (1:5,000; cat no. 7071; Cell Signaling Technology, Inc., Danvers, MA, USA) for 2 h at room temperature. Immunoreactive bands were detected using an ECL detection system (cat no. 6883, Cell Signaling Technology, Inc.) and analyzed with Quantity One 4.0 software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
5
Overexpression and Knockdown of SARS-CoV-2 Nsp11
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The 293 T cells were cultured in 24-well plates and transfected with pcDNA3.1-GFP-nsp11 or pcDNA3.1-GFP and nsp11 siRNA (100 nM) or control siRNA (100 nM) in triplicate, and 36 h later, the cells were lysed with lysing buffer (1 % Nonidet P-40, 0.1 % sodium deoxycholate, 0.1 % SDS, 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM Na3VO4, 2 mM NaF and a protease inhibitor cocktail). The detailed procedure for immunoblots has been described in our previous work [13 (link)]. Briefly, the proteins were separated by 10 % SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Company, Boston, Massachusetts, USA), and then the PVDF membranes were incubated with anti-GFP (Clontech) or anti-actin (Cell Signaling Technology) antibodies. Subsequently the membranes were incubated with appropriate secondary antibodies and were tested by an ECL detection system (Cell Signaling Technology, Boston, USA).
6
Quantitative Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as previously described.40 (link) Briefly, pancreatic tissue was added to RIPA lysis buffer and protease inhibitor for tissue homogenization. Cell lysates were collected by centrifugation at 4°C (12,000 rpm for 15 min). Protein level was quantified using BCA assay. Protein samples (20 μg) were loaded on SDS-PAG of 10% and electrophoresis was performed followed by membrane transfer. The membrane was blocked in Tris-buffered saline tween-20 (TBST) with 5% skimmed milk (for 2 hrs. at room temperature), and then incubated overnight at 4°C with rabbit anti-rat primary antibodies diluted with TBST (LC3 II 1:1000, Cell Signaling technology, Catalog number: 3868). The membranes were then washed and incubated with the secondary antibody (Anti-rabbit secondary antibody conjugated to horseradish peroxidase, DAKO, 1:5000) (for 1.5 hr. at room temperature). Finally, the blot was washed several times, and the chemiluminescent substrate (ECL) detection system (Cell Signaling, Beverly, MA, USA) was applied for visualization. Quantification was performed by Image analysis software for assessing band intensity with β actin as the control reference.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein was extracted from tissues and cells for analysis. The samples were boiled at 100 °C for 5 min. Equal amounts of protein were separated by SDS-PAGE followed by transfer to PVDF membranes (Millipore, USA). The membranes were incubated with primary antibodies overnight at 4 °C. The following primary antibodies: mouse anti-human TRIM44 (ab236422; 1:1,000 dilution; Abcam, Cambridge, UK), rabbit anti-human CCND3 (ab112034, 1:1,000 dilution; Abcam, Cambridge, UK), rabbit anti‐human FOXM1 (ab245309, 1:1,000 dilution, Abcam, Cambridge, UK), rabbit anti-human EZH2 (#4905,1:100 dilution, Cell Signaling Technology), rabbit anti-human CCNE2 (ab226388, 1:1,000 dilution; Abcam, Cambridge, UK), rabbit anti-human BIRC5 (PAB18224, 1:1,000 dilution; Abnova, China), and mouse anti-GADPH (AG019, 1:1,000 dilution, Beyotime) as internal controls. Immunoreactive protein bands were detected using an ECL detection system (Cell Signaling Technology, USA).
8
Dicer Knockdown and PRRSV Infection
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cells of MARC-145 or PAMs were grown in 24-well plates and transfected in triplicate with Dicer siRNA(100 nM) or control siRNA (100nM), and 24 h later, PRRSV (BJ-4 strain) infected the transfected cells at an MOI of 1 or 0.1; then, forty-eight hours later, the cells were dissolved in Nonidet P-40 (1%). The previous work [44 (link)] was described the detailed procedure for immunoblots. Ten percent SDS-PAGE gels were used to separate proteins. Then, the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore Company, Boston, MA, USA), which were then reacted with the proper antibodies and tested by an ECL detection system (Cell Signaling Technology.
9
Protein Extraction and Western Blotting
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted from the cells with 2× Laemmli sample buffer (125 mM Tris, pH 6.8, 4% (w/v) SDS, 20% (v/v) glycerol, 5% (w/v) β-mercaptoethanol), separated by SDS–polyacrylamide gel electrophoresis and transferred onto Immobilon-P membranes (Millipore, Billerica, MA, USA). Blots were incubated with primary antibodies and horseradish peroxidase-conjugated secondary antibodies and developed by the ECL detection system (Cell Signaling Technology, Danvers, MA, USA).
10
Apigenin Inhibits C5aR-Mediated Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Apigenin was purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Monoclonal antibody against C5aR (sc-271949) was supplied by Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Monoclonal antibodies against human PCAF (3378), STAT3 (9139), and β-actin (3700) were from Cell Signaling Technology (Danvers, MA, U.S.A.). Horseradish peroxidase-conjugated anti-mouse IgG (7076) and anti-rabbit IgG (7074), as well as ECL detection system were purchased from Cell Signaling Technology. PVDF membranes were from Millipore (Billerica, MA, U.S.A.). The pcDNA3.1 vector was from Invitrogen. The incision enzymes of HindIII and BamHI as well as T4 DNA ligase were purchased from TaKaRa (Tokyo, Japan). Human C5a was from R&D Systems (Minneapolis, MN, U.S.A.). Cell counting kit-8 (CCK-8) was purchased from Dojindo Laboratories (Kumamoto, Japan). FuGENE®HD was from Promega (Madison, WI, U.S.A.).
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!