HEK-293 cells were plated in six-well dishes and transfected eight hours later with empty or SOX5 expression plasmid (1 μg) and FuGENE6 (3 μl, Promega). The next day, extracts were prepared using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) and tested by western blotting using SOX5 antibody (1:1000) and horseradish peroxidase–conjugated goat anti-rabbit IgG (1:5000, Vector Biolabs). Signals were visualized using ECL Prime Western Blotting Detection Reagents (Amersham). Electrophoretic mobility shift assay (EMSA) was conducted using the same extracts, 10 fmoles [α−32P]-dCTP-labeled 2HMG probe and 1 μg poly(dG-dC).poly(dG-dC), as described.34 (link) Homodimerization was tested in western blots following cell extract incubation for 10 minutes with 0.01% glutaraldehyde.
Ecl prime western blotting detection reagent
Manufactured by Cytiva
Sourced in United States, United Kingdom, Japan, France, Sweden
ECL Prime Western Blotting Detection Reagent is a chemiluminescent substrate used for the detection of proteins in Western blot analysis. It is designed to produce a luminescent signal proportional to the amount of target protein present in the sample.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p, by Merck Group (12 mentions)
Complete protease inhibitor cocktail, by Roche (9 mentions)
Ripa buffer, by Thermo Fisher Scientific (9 mentions)
Pvdf membrane, by Merck Group (8 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (8 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (8 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Roche (6 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Protease inhibitor cocktail, by Merck Group (5 mentions)
Pvdf membrane, by Bio-Rad (5 mentions)
β actin, by Merck Group (5 mentions)
Image lab, by Bio-Rad (5 mentions)
Chemidoc, by Bio-Rad (5 mentions)
Nitrocellulose membrane, by Cytiva (5 mentions)
Nitrocellulose membrane, by GE Healthcare (4 mentions)
Chemidoc mp imager, by Bio-Rad (4 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Santa Cruz Biotechnology (4 mentions)
Fusion fx imager, by Vilber (4 mentions)
Hyperfilm ecl, by Cytiva (4 mentions)
218 protocols using ecl prime western blotting detection reagent
1
SOX5 Protein Expression and Homodimerization
2
Western Blot Analysis of EIF2S1 and Phospho-EIF2S1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All samples were incubated at 95 °C for 10 min, separated on a 4–12% polyacrylamide Bis-Tris protein gel (NP0322BOX, Thermo), and transferred to polyvinylidene fluoride membranes (IPVH00010, Millipore). The membranes were blocked in 10% w/v milk (1706404, Bio-Rad). The blots were probed with 1/1000 v/v EIF2S1 antibody (AHO0802, Invitrogen) or 1/500 v/v Phospho-EIF2S1 (Ser51) antibody (MA5-15133, Invitrogen), followed by 1/10000 v/v sheep anti-mouse IgG (NA931V, Cytiva) or 1/10000 v/v donkey anti-rabbit IgG conjugated to horseradish peroxidase (NA934V, Cytiva). The blots were visualized with ECL Prime Western Blotting Detection Reagents (RPN2232, Amersham) using a Bio-Rad Chemi-Doc MP.
3
Protein Purity Estimation by SDS-PAGE and Western Blot
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein purity estimation was carried out using SDS-PAGE on 4–20% precast acrylamide gels (Biorad 4–20% Mini-Protean TGX Precast Protein Gels, 15-well). Gels were stained with Coomassie Brilliant Blue R-250 Staining solution (Biorad).
Transferring proteins from SDS-gels to PVDF membranes (BioRad) was performed using the BioRad Turbo blotting system (Trans-Blot Turbo Transfer System). After the transfer, membranes were washed three times with PBS-T (PBS, 0.08% Tween-20, pH 7.4) and blocked with 5% BSA in PBS-T. Afterwards, membranes were incubated with the primary anti-DPP3 mAb (murine AK1967, 2 μg/mL in PBS-T with 1% BSA) over night at 4°C on a shacking platform and washed five times. Membranes were incubated with secondary antibody (Anti-mouse IgG, HRP-linked antibody, Cell Signaling Technology, 1:5000 in PBS-T with 1% BSA) at room temperature for 1 hour on a rocking shaker. Afterwards membranes were washed three times. Membranes were developed by incubation with ECL Prime Western Blotting Detection Reagents (Amersham GE Healthcare). Chemiluminescence was detected using the LAS 500 imager system (GE Healthcare).
Transferring proteins from SDS-gels to PVDF membranes (BioRad) was performed using the BioRad Turbo blotting system (Trans-Blot Turbo Transfer System). After the transfer, membranes were washed three times with PBS-T (PBS, 0.08% Tween-20, pH 7.4) and blocked with 5% BSA in PBS-T. Afterwards, membranes were incubated with the primary anti-DPP3 mAb (murine AK1967, 2 μg/mL in PBS-T with 1% BSA) over night at 4°C on a shacking platform and washed five times. Membranes were incubated with secondary antibody (Anti-mouse IgG, HRP-linked antibody, Cell Signaling Technology, 1:5000 in PBS-T with 1% BSA) at room temperature for 1 hour on a rocking shaker. Afterwards membranes were washed three times. Membranes were developed by incubation with ECL Prime Western Blotting Detection Reagents (Amersham GE Healthcare). Chemiluminescence was detected using the LAS 500 imager system (GE Healthcare).
4
Western Blot Analysis of HA-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cell debris was prepared by homogenizing the induced cells, and the protein lysate was prepared using NP-40 buffer (150 mM NaOH, 1.0 % NP-40, 50 mM tris (pH 8.0)) containing protease and phosphatase. Their protein concentrations were quantitated using Quick Start Bradford 1X Dye reagent (Bio-rad). The samples were run on a 15 % SDS-PAGE, transferred to nitrocellulose membranes, and were then blocked with 3 % bovine serum albumin in T-TBS (20 mM Tris, 150 mM NaCl, 0.1 % Tween 20 detergent). The membranes were incubated overnight at 4 ℃ with Anti-HA tag (abcam, 1:500 dilution). The blots were washed and incubated for 1 h at room temperature with the Gt anti-Ms IgG (H+L) secondary antibodies (Invitrogen, 1:5000 dilution). Immunoreactive protein bands were visualized with ECL Prime Western Blotting Detection Reagents (Amersham) and light emission detected by exposing the membrane to X-ray film (AGFA).
5
Western Blotting Protein Analysis Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was carried out as described previously with slight modifications.12 The primary antibodies were: anti‐FLAG M2 (Sigma‐Aldrich, St. Louis, MO, USA), anti‐p‐AKT S473 D9E, anti‐pan AKT, anti‐p‐ERK, anti‐total ERK (Cell Signaling Technology, Danvers, MA, USA), anti‐WDR20 (Bethyl, Montgomery, TX, USA), and anti‐GAPDH (Sigma‐Aldrich). Detection was carried out with ECL Prime Western Blotting Detection Reagents (Amersham Biosciences, Piscataway, NJ, USA) in accordance with the manufacturer's instructions. GAPDH was used as an internal control.
6
DENV2-E Protein Interaction Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Viral stocks were incubated 1:1 v/v with the differentially treated C. sp. Panama culture supernatants, their protein extracts or control buffers for 1 hour at room temperature. For heat inactivation, the protein extract was incubated at 90°C for 1 hour prior to being added to the virus suspension. Samples were then incubated at 85°C for 2 minutes in SDS-containing loading buffer and ran on 4–20% tris-glycine gels under denaturing conditions. Protein bands were dry-transferred to a nitrocellulose membrane, followed by overnight blocking at 4°C in 5% skim milk 0.1% Tween 20 PBS. Membranes were probed with an anti-DENV2-E rabbit polyclonal (1:2,000) followed by anti-rabbit HRP-linked donkey (1:20,000, Amerhsam) antibodies for 1 hour each, with abundant wash with 0.1% Tween 20 PBS after each probing. Chemiluminescence signals were detected using Amersham ECL Prime Western Blotting detection reagents.
7
CXCR4 Expression Analysis in HEp Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
P-HEp3, Lu-HEp3, and BM-HEp3 cells were washed once in ice-cold PBS and then lysed by the addition of CelLytic M Cell Lysis/Extraction Reagent (Sigma) containing freshly added protease inhibitor cocktail (Sigma), 50 mM NaF and 1 mM Na3VO4. Supernatants were stored at −80 °C until use. The protein concentration was determined using a BCA kit (Pierce Chemical, Rockford, IL). Equal amounts of protein were fractionated via SDS–PAGE and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% non-fat dried milk and 0.1% Tween 20 (Sigma) in PBS (pH 7.4) and were then incubated overnight at 4 °C with antibodies against CXCR4 (1: 500; Abcam) and β-actin (1: 5,000; Sigma) in 5% BSA (Sigma) and 0.1% Tween 20 in PBS (pH 7.4). After the membranes were washed, they were incubated for 1 hour in horseradish peroxidase-conjugated secondary antibodies. After a washing, specific protein bands were detected by using ECL Prime Western Blotting Detection Reagents (Amersham Life Science, Arlington Heights, IL) according to the manufacturer's instructions.
8
Western Blot Analysis of Immune Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were washed once in ice-cold PBS and then lysed by adding CelLytic M Cell Lysis/Extraction Reagent (Sigma) containing freshly added protease inhibitor cocktail (Sigma), 50 mM NaF, and 1 mM Na3VO4. Supernatants were stored at −80°C until use. Equal amounts of protein were fractionated via sodium dodecyl sulfate−polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked with 5% non-fat dried milk and 0.1% Tween 20 (Sigma) in PBS (pH 7.4) and were then incubated overnight at 4°C with antibodies against IL-6 (1:2000), CYLD (1:600), FLAG (1:3800), or β-actin (1:5000) in 5% bovine serum albumin (Sigma) and 0.1% Tween 20 in PBS (pH 7.4). After the membranes were washed in 0.1% Tween 20 in PBS (pH 7.4), they were incubated for 1 h in horseradish peroxidase-conjugated secondary antibodies for 1 h. After another washing, specific protein bands were detected by using ECL Prime Western Blotting Detection Reagents (Amersham Life Science, Arlington Heights, IL, USA), according to the manufacturer's instructions.
9
Quantitative Western Blotting of HIF-1α and Erk Signaling
10
DAPK1 Expression Analysis in Diverse Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MEC1, MEC2, 232B4, CLL-AAT and WAC3CD5+ cells were harvested and then lysed in RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.2% SDS, 1% Triton X-100, 2 mM EDTA). Protein lysates were resolved on 6% SDS-PAGE and electrotransferred onto a 0.2 μm nitrocellulose membrane (Bio-Rad, Hercules, CA). The membranes were blocked and incubated with anti-DAPK1 (1:1000; Sigma-Aldrich, USA) or anti-actin (1:5000; Sigma-Aldrich, USA) primary antibody at 4°C overnight. Then membranes were washed three times and incubated with anti-rabbit horseradish peroxidase conjugate secondary antibody at room temperature for 1 hour. Protein signals were detected by ECL Prime Western blotting detection reagents (Amersham Biosciences, Buckinghamshire, UK).
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!