IB was carried out as described previously19 (link),29 (link). Briefly, cells were grown in six-well tissue culture plates in medium supplemented with 10% FBS for 48 h and transfected with a siRNA pool targeting ELF1 or MAZ and an expression vector bearing the HA-tagged ELF1 or the MAZ cDNA for 48 h. Cells were collected and protein isolation was performed using the NE-PER protein extraction kit (Thermo-Fisher). Protein concentration was determined using Bradford Proteins Assay (Bio-Rad). Nuclear extracts were subjected to denaturing SDS-PAGE, transferred to a membrane and proteins were probed with an antibody specific to ELF1 or MAZ followed by a secondary antibody conjugated with the horseradish peroxidase (Advansta). The membranes were then re-probed with the HA antibody (Abcam, ab9110) and subsequently with an HDAC1 antibody (Abcam, ab19845) as a loading control. Images were developed using the ECL-Substrate (Advansta) and captured with ChemiDoc Imaging System (Bio-Rad).
Ecl substrate
Manufactured by Advansta
Sourced in United States
ECL substrate is a chemiluminescent detection reagent used in Western blotting analysis to visualize and quantify proteins. It produces a luminescent signal when catalyzed by the horseradish peroxidase (HRP) enzyme, which is commonly used to label antibodies in immunodetection procedures.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti mouse secondary antibody, by Promega (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Hrp conjugated secondary antibody, by Abcam (2 mentions)
Tanon 4200sf, by Advansta (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Magna lyser, by Roche (2 mentions)
Ab19845, by Abcam (1 mentions)
Horseradish peroxidase, by Advansta (1 mentions)
Ab9110, by Abcam (1 mentions)
Ne per protein extraction kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc imaging system, by Bio-Rad (1 mentions)
Sample buffer, by GenScript (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Imagequant, by GE Healthcare (1 mentions)
Versadoc system, by Bio-Rad (1 mentions)
Immobilon p pvdf membrane, by Merck Group (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
13 protocols using ecl substrate
1
Characterization of ELF1 and MAZ Proteins
2
Western Blot Analysis of M1/M2 Macrophage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Macrophage pellets were re-suspended in 30 μl of RIPA buffer (Beyotime, China). After incubation on ice for 1 h, the lysates were centrifuged at 12,000 rpm for 30 min at 4°C. The supernatant was mixed with sample buffer (Genscript, China) and incubated in boiling water for 10 min. The total cellular protein (30 μg/well) was separated by 10% SDS-PAGE. The separated proteins were transferred onto polyvinylidene difluoride membranes (PVDF, Millipore), which were then blocked overnight with 5% skim milk in Tris-buffered saline containing 0.1% Tween-20 (TBST). The membranes were respectively incubated with primary antibodies to detect Arg-1 (1: 1000 Cell Signaling Technology) and MRC-1 (1: 1000 Proteintech). The housekeeping gene encoding beta-actin (1: 5000 Thermo Fisher Scientific) was used as an internal control. Finally, the target protein bands were visualized by ECL substrate (Advansta, China), and images were collected by the ChemiDoc XRS+ system (Bio-Rad, Inc.).
3
SDS-PAGE and Immunoblotting of Viral Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 10 or 4–12% gradient acrylamide gels. Generally, gels were ran for 1 h at 160 V. Total proteins were visualized by Coomassie brilliant blue staining for 1 h and destained until the background became transparent.
For immunoblotting, after SDS-PAGE, proteins were transferred onto polyvinylidene fluoride membrane (Millipore) for 2 h 30 min at 300 mA. Membranes were blocked in 5% non-fat milk (dissolved in TBST buffer containing 25 mM Tris, 250 mM NaCl, 0.1% Tween-20, pH 7.4) for at least 2 h at room temperature. For detection of soluble gp350-ECD123-6His, primary mouse anti-His antibody (Abmart) was diluted 1:2,000 and incubated with membrane at 4°C overnight following incubation with secondary goat anti-mouse antibody (1:20,000, Promega) conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. For detection of Fc fusion proteins, the membranes were incubated directly with secondary goat anti-mouse antibody (1:20,000) for detection using ECL substrate (Advansta).
For immunoblotting, after SDS-PAGE, proteins were transferred onto polyvinylidene fluoride membrane (Millipore) for 2 h 30 min at 300 mA. Membranes were blocked in 5% non-fat milk (dissolved in TBST buffer containing 25 mM Tris, 250 mM NaCl, 0.1% Tween-20, pH 7.4) for at least 2 h at room temperature. For detection of soluble gp350-ECD123-6His, primary mouse anti-His antibody (Abmart) was diluted 1:2,000 and incubated with membrane at 4°C overnight following incubation with secondary goat anti-mouse antibody (1:20,000, Promega) conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. For detection of Fc fusion proteins, the membranes were incubated directly with secondary goat anti-mouse antibody (1:20,000) for detection using ECL substrate (Advansta).
4
Phospho-Protein Analysis of Tissue Explants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For Western blot analyses, tissue explants were collected and immediately stored at−80ºC after a 15-min incubation under different experimental conditions. Tissue explants (100 mg/ ml, n = 5–9) were homogenized and processed for Western blot as previously described by Matafome (Matafome et al., 2012 (link)). Primary antibodies to the phosphorylated forms of insulin receptor (InsR, Tyr972, ab5678), protein kinase B (PKB), also known as Akt (Akt, Ser473, #4058S), adenosine monophosphate kinase (AMPK, Thr172, #2535S), hormone sensitive lipase (HSL, Ser563, #4139S), acetyl CoA carboxylase (ACC, Ser79, #11818P), and ATP citrate lyase (ACL, Ser455 #4331P) were obtained from Abcam (Cambridge, United Kingdom) or Cell Signalling Technology (Danvers, United States). Calnexin (AB0041-200) from SICGEN (Cantanhede, Portugal) was used as the loading control. Secondary antibodies of anti-rabbit and anti-goat samples were obtained from Bio-Rad (Spain) and Invitrogen or Thermo Fisher Scientific (United States), respectively. Membranes were revealed using an ECL substrate (Advansta, CA, United States) in a VersaDoc system (Bio-Rad, United States) and analyzed with ImageQuant® (Molecular Dynamics, United States).
5
Larval Fat Body Protein Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Early third instar larval fat bodies were dissected in PBS and immediately homogenized in lysis buffer. Protein samples were separated in 10% poly-acrylamide gel and transferred to 0.45 μm Immobilon-P PVDF membrane (Millipore). The membrane was blocked for 1 h at RT in TBST containing 5% skim-milk and incubated overnight at 4°C with anti-Akt (1:1000, Cell Signaling Technology, #9272) and anti-β-actin (1:1000, Cell Signaling Technology, #4967) antibodies. All primary antibodies were diluted with TBST containing 5% BSA and 0.1% sodium azide. After washing, the membrane was incubated in TBST containing 5% skim-milk and HRP-conjugated secondary antibody (1:10000, Abcam, ab6721) for 1 h at RT. HRP reaction was generated by ECL substrate (Advansta) and detected by a chemiluminescence imaging system (UVITEC).
6
Western Blot Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in RIPA lysis buffer (Beyotime, China) supplemented with PMSF (Beyotime, China). Cell lysates were cleared by centrifugation at 12000 rpm at 4°C for 20 min and collected. Protein concentration was determined by the BCA protein assay (Beyotime, China). Proteins were separated by SDS/PAGE (10% gel) and transferred on to PVDF membranes (Immobilon-P, Millipore, Germany). Membranes were blotted with the appropriate primary antibodies overnight at 4°C and then secondary antibodies for 1 h at room temperature. The antigen–antibody complexes were detected by ECL substrate (Advansta, California, U.S.A.).
7
Gingival Tissue Protein Extraction
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gingival tissues were isolated and homogenized in ice-cold lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.5% Nonidet P-40, 0.1% SDS, and 0.5 mM EDTA) supplemented with a protease inhibitor mixture (Sigma). After lysate incubation for 30 min on ice, tissue debris were removed by centrifugation (15,000 × g for 30 min at 4°C), and protein concentration in the supernatant was determined using the Pierce BSA Protein Assay kit (Thermo Scientific). Samples of 5–20 µg total protein were loaded onto 10% acrylamide gel and subject to SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore), the membrane was blocked in 5% skim milk for 30 min at room temperature, and reacted with primary antibody overnight at 4°C. The primary antibodies used are rabbit anti–TGFβ1 (Abcam), mouse anti–BMP-7 (Abcam), and rabbit polyclonal anti-Actin (I-19; Santa Cruz Biotechnology). The membrane was then washed three times in 1× TBST for 10 min at room temperature, incubated with secondary HRP-conjugated antibody (Abcam) in blocking buffer for 1 h at room temperature and washed three times in TBST before the blots were reacted with ECL substrate (Western blot detection kit; Advansta). Images were captured using a ChemiDoc MP Image System (Bio-Rad), and band quantification was done using ImageJ (National Institutes of Health).
9
Quantifying P3H2 Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot experiments were performed following standard procedures. First, 50–100 µg of total protein extracts were run on 10% SDS-polyacrylamide gel and transferred to a PVDF membrane (Millipore, Burlington Middlesex County, MA, USA) and incubated with antibodies against human P3H2 (SIGMA, 1:1000). Protein loading was assessed using antibody anti β-Tubulin (Santa Cruz Biotechnology, Dallas, TX, USA, 1:2000) or anti-Vinculin (Cell Signaling, Danvers, MA, USA 1:10,000). The secondary antibodies were from DAKO, Santa Clara, CA, USA, (1:10,000). The signals were visualized by chemiluminescence using ECL substrate (Advansta, Menlo Park, CA, USA).
10
Visualization of Viral Protein Profiles
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein samples were subjected to 12% SDS-PAGE gels for 1 h at 160 V and the total proteins were visualized by coomassie brilliant blue staining.
For WB, after SDS-PAGE, proteins were transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk in TBST buffer (25 mM Tris, 250 mM NaCl, 0.1% Triton-20, pH 7.4) for 2 h at room temperature. To detect the HBc149 antigen, mAb 11H10 (gift from Prof. Xia of Xiamen University) was used (1:2,000 dilution). For detection of gp350 epitopes, an antiserum prepared in our lab was used (1:1,000 dilution). The membranes were inoculated with diluted antibodies at 4°C overnight. After three washes with TBST, membranes were incubated with a secondary goat anti-mouse antibody (1:10,000, Promega) conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. ECL substrate was used for detection (Advansta).
For WB, after SDS-PAGE, proteins were transferred onto polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk in TBST buffer (25 mM Tris, 250 mM NaCl, 0.1% Triton-20, pH 7.4) for 2 h at room temperature. To detect the HBc149 antigen, mAb 11H10 (gift from Prof. Xia of Xiamen University) was used (1:2,000 dilution). For detection of gp350 epitopes, an antiserum prepared in our lab was used (1:1,000 dilution). The membranes were inoculated with diluted antibodies at 4°C overnight. After three washes with TBST, membranes were incubated with a secondary goat anti-mouse antibody (1:10,000, Promega) conjugated with horseradish peroxidase (HRP) for 1 h at room temperature. ECL substrate was used for detection (Advansta).
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!