The protocol used for western blot has been previously described in detail [47 (link)]. In brief, Cell lysates were prepared with RIPA buffer (150 mM NaCl, 20 mM Tris-Cl pH7.5, 1% Triton X-100, 1% NP40, 0.1% SDS, 0.5% deoxycholate) containing freshly added proteinase and phosphatase inhibitors (Bionovas, Toronto, Canada). Protein concentrations were measured with the Bradford method. Each sample (100 μg) was subjected to electrophoresis in 10% SDS-polyacrylamide gels and subsequently transferred onto nitrocellulose membranes. All experiments were conducted using commercially available antibodies and corresponding horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology). The specific rabbit polyclonal antibody against phospho-ETS1-Thr265, Ser269 was produced by ABclonal (Woburn, MA, USA) using the following immunogen: CDRL(Tp)QSW(Sp)SQSS (phospho-ETS1-Thr265, Ser269). After immunization for five times in two months, the antibodies were purified by antigen affinity chromatography. Chemiluminescence reagents were obtained from Millipore. The signal intensity of the autoradiograms was quantified using the ImageJ software (https://imagej.nih.gov/ij/ ) after normalization for the corresponding actin intensity. The antibodies used for western blot are shown in Supplementary Table 2 .
Horseradish peroxidase conjugated antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Horseradish peroxidase-conjugated antibodies are enzyme-linked antibodies used in various immunoassays and detection techniques. They contain an antibody molecule coupled to the enzyme horseradish peroxidase, which can catalyze a colorimetric reaction for signal amplification and visualization.
Automatically generated - may contain errors
Lab products found in correlation
Gapdh, by Santa Cruz Biotechnology (4 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (4 mentions)
Chemiluminescence reagent, by Merck Group (3 mentions)
Supersignal west dura chemiluminescent substrate, by Thermo Fisher Scientific (3 mentions)
Rabbit anti phospho akt p ser473 antibody, by Cell Signaling Technology (3 mentions)
Ecl reagent, by Thermo Fisher Scientific (3 mentions)
Ubiquitin, by Cell Signaling Technology (3 mentions)
Halotag, by Promega (3 mentions)
Actin, by Santa Cruz Biotechnology (3 mentions)
Bicinchoninic acid kit bca kit, by Merck Group (2 mentions)
Rabbit polyclonal antibody to β actin, by Abcam (2 mentions)
Bicinchoninic acid method, by Thermo Fisher Scientific (2 mentions)
Keap1, by ABclonal (2 mentions)
Phosphorylated ser349 p62, by Cell Signaling Technology (2 mentions)
Enhanced chemiluminescence reagent, by Merck Group (2 mentions)
Calmodulin binding peptide tag cbp, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Bio-Rad (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
54 protocols using horseradish peroxidase conjugated antibody
1
Western Blot Analysis of Phospho-ETS1
2
Western Blot Analysis of Signaling Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded in Petri dishes with the appropriate medium and grown to a confluency of 80%. Before cell lysis, Petri dishes were kept on ice and washed twice with ice–cold PBS. Cells were lysed with RIPA Buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP–40, 1% sodium deoxycholate, and 0.1% SDS) containing the following protease inhibitors: 2 mg/mL aprotinin, 1 mM Na orthovanadate, 0.1 mM PMSF, and 10 mM sodium fluoride. Lysates were centrifuged at 4 °C for 10 min at 12,000× g. Protein concentrations were determined using a Bicinchoninic Acid Kit (BCA kit, Sigma) following the manufacturer’s instructions. A quantity of 50 µg of the sample was resuspended in SDS sample buffer, heated at 95 °C for 5 min, and separated on 10% pre–cast SDS gel (Biorad). Polyvinylidene fluoride membranes were properly blocked and then incubated overnight with ERK1/2 (1:1000, #9102, Cell Signaling), phospho–ERK1/2 Thr402/Tyr204 (1:1000, #9101, Cell Signaling), AKT (1:3000, #9272, Cell Signaling), and Phospho–Akt–Ser473 (1:2000, #4508, Cell Signaling) antibodies. The membrane was then washed with TBS containing 0.1% Tween 20 and incubated with the appropriate horseradish–peroxidase–conjugated antibodies (Santa Cruz). Chemiluminescence assays were conducted using the SuperSignal West Dura chemiluminescent substrate (Thermo Fischer Scientific).
3
Quantitative Analysis of Cytoskeletal Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins were extracted after 4 h and 24 h using a RIPA lysis buffer and the concentrations were determined using Bio-Rad protein assay reagent (Bio-Rad Laboratories s.r.l.). Equal amounts of protein (40 µg) were loaded onto a polyacrylamide gel for SDS-PAGE and transferred to a nitrocellulose membrane. The filters were incubated overnight at 4 °C with antibodies against elastin (1:250), tubulin (1:500), and actin (1:1000). The membranes were washed three times for 10 min and incubated with anti-rabbit (1:10,000), anti-mouse (1:5000), or anti-goat (1:5000) horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Protein levels were normalized with respect to the signal of anti-actin polyclonal antibody. Blots were developed using the ECL system (Enhanced Chemiluminescence) (Amersham Biosciences, Little Chalfont Buckinghamshire United Kingdom) according to the manufacturer’s protocol. Densitometric analyses were performed using a Gel Doc 2000 UV System and the Gel Doc EZ Imager (Quantity One software, System ID 4020DABD, Bio-Rad Laboratories s.r.l.).
4
Immunoblot Analysis of Glioma IDH Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein levels of F98 IDH1 –WT/–R132H, IDH2 –WT/–R172K, and Mock gliomas were evaluated by immunoblot analysis. Cells were lysed in ice–cold lysis buffer composed of 20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM β–glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin, and protease inhibitor cocktail (Sigma, MO, USA), and the concentration of lysate protein was evaluated with the bicinchoninic acid method (Pierce Biotechnology, Rockford, IL, USA). Approximately 50 μg of protein was loaded in each lane of a polyacrylamide denaturing gel for electrophoresis. After electrophoresis, the protein was transferred to nitrocellulose membranes for blotting. We used a rat monoclonal antibody to IDH1 (Dianova, Hamburg, Germany), a mouse monoclonal antibody to IDH1–R132H (Dianova), a rabbit polyclonal antibody to IDH2 (Proteintech, Chicago, IL, USA), a mouse monoclonal antibody to IDH2–R172K (NewEast Biosciences, King of Prussia, PA, USA), and a rabbit polyclonal antibody to β–actin (Abcam, Cambridge, UK). Primary antibodies were detected by horseradish peroxidase–conjugated antibodies (Santa Cruz Biotechnology, Paso Pobles, CA, USA).
5
Western Blot and Immunoprecipitation Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were subjected to 8–12% SDS-PAGE and transferred onto a PVDF membrane (Bio-Rad Laboratories). Membranes were blocked in 5% milk in tris-buffered saline, containing 0.05% Tween 20 for 1.5 h at room temperature. Membranes were then incubated with different primary antibodies overnight at 4 °C. The membranes were washed in TBS-T and incubated with secondary horseradish peroxidase-conjugated antibodies (Santa Cruz, CA, USA; 1:5000) for 2 h at room temperature. Blots were then visualized using enhanced chemiluminescence reagents (Bio-Rad Laboratories). The density of the immunoreactive bands was analyzed using Image J software (NIH, Bethesda, MD, USA).
Nuclear proteins, where indicated, were prepared using a cytoplasmic and nuclear protein extraction kit (KeyGEN, Nanjing, China). Levels of nuclear p65 subunit were probed using Western blotting.
For immunoprecipitation studies, TAK1 was co-precipitated with FAK from macrophages to detect the association of TAK1 with FAK. Cell extracts were incubated with anti-FAK antibody for 1 h and then precipitated with protein G-Sepharose beads at 4 °C overnight. TAK1 levels were detected by immunoblotting.
Nuclear proteins, where indicated, were prepared using a cytoplasmic and nuclear protein extraction kit (KeyGEN, Nanjing, China). Levels of nuclear p65 subunit were probed using Western blotting.
For immunoprecipitation studies, TAK1 was co-precipitated with FAK from macrophages to detect the association of TAK1 with FAK. Cell extracts were incubated with anti-FAK antibody for 1 h and then precipitated with protein G-Sepharose beads at 4 °C overnight. TAK1 levels were detected by immunoblotting.
6
Protein Detection and Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples were prepared from the cells lysed with CelLytic M cell lysis reagent (Sigma-Aldrich) or NE-PER reagent (Thermo Scientific) containing a complete protease inhibitor cocktail (Roche Applied Science). Total proteins were separated by electrophoresis on 4% to 12% gradient PAGE gels (Bio-Rad) and transferred to polyvinylidine difluoride membrane (GE Healthcare). Proteins of interest were detected by incubating membrane with horseradish peroxidase–conjugated antibodies (Santa Cruz) and visualized with enhanced chemiluminescence (GE Healthcare) or SuperSignal West Dura Chemiluminescent Substrate (Thermo Scientific). The following antibodies were used: rabbit polyclonal SUV39H2 antibody (ab71683; Abcam), rabbit polyclonal H3 antibody (ab1791; Abcam), rabbit polyclonal H3K9me3 antibody (ab8898; Abcam), caspase-3 and cleaved caspase-3 (Cell Signaling), anti-hemagglutinin (HA) high affinity (clone 3F10; Roche Life Science), and mouse monoclonal β-actin (AC-15; Sigma-Aldrich).
7
Western Blot Analysis of Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot analysis was performed to investigate protein expression. Cells cultured in 6-well plates were washed with PBS twice and harvested on the floor of the plate. Cells were lysed using radioimmunoprecipitation assay lysis buffer (Sigma-Aldrich; Merck KGaA) supplemented with a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Cell lysates were centrifuged at 13,000 × g for 15 min at 4°C and protein concentration was determined with a BCA assay. Samples (10 µg protein) were loaded onto 10% SDS-PAGE gels. Gel proteins were electrophoretically transferred to polyvinylidene difluoride membranes (PVDF; Thermo Fisher Scientific, Inc.). Membranes were blocked with 3% non-fat dried milk in PBS at 4°C overnight. Subsequently, PVDF membranes were incubated with specific primary antibody solutions against antigens for 24 h at 4°C. Detection of the primary antibodies was performed using secondary, horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Inc.). Details of the antibodies used have been provided earlier in the manuscript. Finally, the antigen-antibody complexes were visualized by a Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific, Inc.; cat. no: 32106) and quantitated using the Versa DOC system and Quantity One software (#1709600; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
8
Western Blot Analysis of ROR2 and Akt
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Subconfluent cells were washed twice with PBS, and then lysed with ice-cold RIPA lysis buffer (50 mmol/L Tris, 150 mmol/L NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mmol/L sodium orthovanadate, 1 mmol/L sodium fluoride, 1 mmol/L EDTA, 1 mmol/L PMSF, and 1% cocktail of protease inhibitors) (pH7.4). The lysates were then clarified by centrifugating at 12,000g for 20 min at 4 °C. The protein extracts were separated by SDS-PAGE. The immunoblotting procedure was performed as described [16 (link)] and the following antibodies were used: rabbit anti-ROR2 antibody, mouse anti-GAPDH antibody (Proteintech, Wuhan, China), rabbit anti-Akt antibody, rabbit anti-phospho-Akt (p-Ser473) antibody (Cell Signaling Technology, Danvers, MA). Protein bands were detected by incubating with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology) and visualized with ECL reagent (Thermo Scientific, Rockford, IL). The gray values were taken by Tanon imaging analysis system (Tanon, Shanghai, China).
9
Western Blot Analysis of EMT Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell lysates were harvested from A549 cells, and proteins were resolved by SDS-PAGE and electro transferred to PVDF membranes where primary antibodies against E-cadherin (1:2000; #610181, BD Biosciences, San Jose, CA, USA), N-cadherin (1:1000; #610921, BD Biosciences, USA), and vimentin (1:500; sc-6260, Santa Cruz Biotechnology, Dallas, TX, USA) were used. Corresponding secondary horseradish peroxidase-conjugated antibodies (1:2000; Santa Cruz Biotechnology, Dallas, TX, USA) were used to detect primary antibodies on the membrane. Membranes were developed using BioRad Clarity Western ECL Substrate reagent in BioRad ChemiDoc imaging system (BioRad, Carlbad CA, USA). Protein levels were quantified by densitometry using Fiji open-source software (Image J version 1.52p; National Institutes of Health, Bethesda, MD, USA). Whole blots can be found in the Supplementary Materials (Figure S6 ).
10
Western Blot Analysis of Apoptosis and Metalloproteinases
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-cell extracts were prepared from A375 cells treated with NAP-HBTA 100 μM or from melanoma tissue homogenate, as previously described (Panza et al., 2015 (link)). The protein concentration was measured by the Bradford method (Bio-Rad, Milan, Italy). Equal amounts of protein (40 μg/sample) were separated by SDS-PAGE and blotted onto nitrocellulose membranes (Trans-Blot Turbo Transfer Starter System, Biorad). The membranes were blocked for 2 h in 5% low-fat milk in PBS with 0.1% Tween 20 (PBST) at room temperature. Then the filters were incubated with the following primary antibodies: caspase 3 and PARP (Cell Signaling, United States; diluted 1:1000), MMP-2 and MMP-13 (Santa Cruz Biotechnology, Santa Cruz, CA, United States; diluted 1:1000), overnight at 4°C. The membranes were washed three times with PBST and then incubated with horseradish peroxidase-conjugated antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, United States; diluted 1:2000) for 2 h at room temperature. The immune complexes were visualized by the ECL chemiluminescence method and acquired by the Image Quant 400 system (GE Healthcare).
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!