Nitrocellulose membranes were blocked in 3% ECL™ Blocking Agent (GE Healthcare, RPN418) in TBST at room temperature for 1 h, then probed with antibodies against eIF4G1 (1:5000), eIF3b (1:5000), eIF3d (1:5000), GADPH (1:5000), eIF4E-BP1 (1:1000), phosphorylated eIF2α (1:500) and detected by chemiluminescence using corresponding anti-rabbit or anti-mouse antibodies at 1:25000 dilution. Incubation with primary and secondary antibodies was also performed in 3% blocking reagent in TBST under the same conditions. Antibodies bound to eIF4G2 and GADPH were visualized with an enhanced chemiluminescence detection kit (ECL™ Prime Western Blotting System, GE Healthcare, RPN2232). eIF4G1, eIF3b, eIF4E-BP1, phosphorylated 4E-BP1 and phoshorylated eIF2α were detected with SuperSignal™ West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific, 34095). Note that for phospho-eIF4E-BP1 detection, the membrane was blocked in 5% BSA in TBST at room temperature for 1 h, incubation with primary antibodies (1:2000) was performed overnight at 4°C in 5% BSA/TBST, corresponding secondary antibodies (1:25 000) were also diluted in 5% BSA/TBST and visualization was performed with ECL™ Prime Western Blotting System (GE Healthcare). The images were captured using ChemiDoc XRS+ with Image Lab™ 3.0 software for image processing and quantification (Bio-Rad).
Ecl prime western blotting system
Manufactured by GE Healthcare
Sourced in United States, United Kingdom
The ECL Prime Western Blotting System is a laboratory equipment used for the detection and analysis of proteins in a Western blot procedure. It provides a sensitive and reliable method for the visualization of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Amersham imager 600, by GE Healthcare (5 mentions)
Trans blot turbo transfer system, by Bio-Rad (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Nitrocellulose membrane, by GE Healthcare (3 mentions)
Polyvinylidene difluoride membrane, by Merck Group (3 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Mouse anti α actin, by Cell Signaling Technology (2 mentions)
Rabbit anti α actin, by Merck Group (2 mentions)
Rabbit anti rig 1 d14g6, by Cell Signaling Technology (2 mentions)
Trans blot turbo, by Bio-Rad (2 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs imaging system, by Bio-Rad (2 mentions)
Gapdh, by Abcam (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (2 mentions)
Anti flag m2 affinity gel, by Merck Group (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Anti myc, by Proteintech (2 mentions)
64 protocols using ecl prime western blotting system
1
Quantitative Western Blot Analysis of eIF Proteins
2
Comprehensive Immunoblotting Techniques
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Immunoblotting was conducted as described previously using ECL™ Prime Western Blotting System (GE Healthcare) [9 (link)]. The primary antibodies used included: Akt (pan) (C67E7) Rabbit mAb #4691, Phospho-Akt (Thr308) (C31E5E) Rabbit mAb #2965, Phospho-p70 S6 Kinase (Thr389) (108D2) Rabbit mAb #9234, p70 S6 Kinase Antibody #9202, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (20G11) Rabbit mAb #4376, p44/42 MAPK (Erk1/2) Antibody #9102, Phospho-Rb (Ser780) (D59B7) Rabbit mAb #8180,GAPDH (14C10) Rabbit mAb #2118, Cyclin D1 (92G2) Rabbit mAb #2978, CDK2 (78B2) Rabbit mAb #2546, Phospho-CDK2 (Thr160) Antibody #2561, Keratin 7 (D1E4) Rabbit mAb #4465 from Cell Signaling Technology, Purified Mouse Anti-Human Retinoblastoma Protein Clone G3–245 (RUO) #554136(BD Pharmingen™), Recombinant Anti-Uroplakin Ia antibody [EPR15498] (ab185970) and Recombinant Anti-NuMA antibody [EP3976] (ab109262) from Abcam, Anti-Cas9, clone 7A9 (Cat.#MAC133) from EMD Millipore Corporation. Secondary HRPO conjugated antibodies were purchased from Dianova.
3
Western Blot Analysis of ALKBH4
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Cell lysates were resolved on 10% SDS-polyacrylamide gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane (Merck Millipore). Membranes were blocked with 5% skim milk (Morinaga Milk Industry) at room temperature (20–25 °C) for 30 min and subsequently incubated overnight with anti-ALKBH4 antibody (NBP2-14737; Novus Biologicals) or anti-β-tubulin antibody (T4026; Sigma-Aldrich) at 4 °C. This was followed by incubation with horseradish peroxidase–conjugated anti-rabbit immunoglobulin G or anti-mouse immunoglobulin G (Santa Cruz Biotechnology) for 1 h. Bound horseradish peroxidase conjugates were visualized using the ECL Prime Western Blotting System (GE Healthcare) and captured using Amersham Imager 680 (GE Healthcare).
4
CRISPR-Cas9 Expression Monitoring in Mycobacteria
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After transformation of M. smegmatis and BCG with pKLM-CRISPR-lysA(x), kanamycin-resistant colonies were recovered and cultured in 5 mL of MB7H9 and MB7H9-OADC, respectively. These cultures were used as pre-inoculum in a fresh culture starting at OD600 0.1. After 2 h (M. smegmatis) or 24 h (BCG) of incubation, tetracycline was added to induce expression of Cas9, and the culture was maintained at 37°C for 4-24 h (M. smegmatis) or 24-120 h (BCG). After the induction, bacteria were lysed by sonication using an ultrasonic processor GE100 (GE Healthcare, Chicago, IL, USA) and protein extracts separated by SDS-PAGE (BioRad, Hercules, CA, UK), transferred to PVDF membranes (GE Healthcare) and blocked with 5% non-fat dry milk at 4°C for 16 h. The membrane was probed using monoclonal anti-Cas9 antibodies (7A9-3A3, 1:1,000) (Santa Cruz Biotechnology, Dallas, TX, USA) incubated for 90 min and anti-mouse IgG conjugated with peroxidase (A6782, 1:1,000) (Sigma-Aldrich®) for 60 min. E. coli DH5α transformed with pCas (20 (link)) was used as positive control for Cas9 expression. Chemiluminescent signal was developed using ECL Prime Western Blotting System (GE Healthcare) and images acquired with the LAS4000 digital imaging system (GE Healthcare).
5
Quantitative Western Blot Analysis
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Fifty nanograms of tPA (Actilyse®), tPA-HaloTag® (coming from tPA-HaloTag®-transfected HEK cells) were loaded in 10% polyacrylamide gel. Polyacrylamide gels were then transferred on a PVDF membrane and immunoblotted with the primary antibodies. After incubation with the secondary antibodies, proteins were visualized with an enhanced chemiluminescence western blot detection reagent (ECL Prime Western Blotting System; GE Healthcare; RPN2232) using ImageQuant LAS 4000 Camera (GE Healthcare, Chicago, IL, USA).
6
Western Blot Protein Analysis
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For western blots 2 × 106 cells were resuspended in 200 μl 2x Laemmli Sample Buffer (0.125 M Tris HCl, 20% glycerol, 4% SDS and 0.002% Bromphenol Blue). Lysates were homogenized through a 23 gauge needle and denatured at 70 °C for 10 min. Between 18 and 25 μl of protein lysate was loaded on 12% SDS-PAGE and transferred onto a PVDF membrane (GE Healthcare Amersham™ Hybond™). After incubation with the specific primary antibody (see Supplementary Information ), the membranes were incubated with appropriate secondary peroxidase-conjugated antibodies. For detection ECL™ Prime Western Blotting System (GE Healthcare, Chicago, IL, USA) and the Gel Logic 1500 imaging system analyzed with Kodak Molecular Imaging Software (Version 5.0) was used.
7
Western Blot Analysis of SNX9 and Fls
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Samples of either Xenopus egg extracts or purified recombinant SNX9 were run on 4–20% mini-PROTEAN precast protein gels (Biorad) and transferred to nitrocellulose membrane using an iblot 2 dry blotting system. Western blots using ScFvs were blocked in 10% milk/PBST, incubated with 4.5 µg/ml ScFv in 1% milk/PBST for 1 h at room temperature, washed with PBST, and then incubated overnight with a 1/1,000 dilution in 1% milk/PBST of either rabbit anti-his antibody (Abcam, ab9108) or mouse anti-myc antibody (Roche, 11667149001). After washing the membranes, tertiary incubations were performed with a 1/10,000 dilution in 1% milk/PBST of either anti-rabbit or anti-mouse 800 CW antibody (LI-COR Biosciences, 926–32210 and 926–32211), washed, and imaged using an Odyssey Sa Reader (LI-COR Biosciences). Western blots using Fls IgGs were blocked in 5% milk/1 × TBS + 0.1% Tween. Antibody incubations were performed in antibody diluted in 5% milk/1 × TBS + 0.1% Tween. Fls IgGs were diluted to 2.5 µg/ml and the secondary mouse anti-Human IgG1-HRP antibody (Thermo Fisher Scientific, A-10648) used at 1/500 dilution. HRP was detected using the ECL Prime Western blotting system (GE Healthcare, GERPN2232).
8
Western Blot Analysis of Rg3 Signaling
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Western blot analysis was performed as previously described [19 (link)]. Briefly, MPBAs were treated with the indicated doses of Rg3 for two days. Total proteins were extracted using lysis buffer containing protease and phosphatase inhibitors, followed by processing for SDS-PAGE. The blots were incubated with the following primary antibodies as appropriate: PPARγ, perilipin, ERO1L, Phospho-eIF2α, CHOP, Phospho-Akt, total Akt, cleaved caspase3, total caspase3, and β-actin (all antibodies purchased from Cell Signaling Technology). An ECL Prime Western Blotting System (GE Healthcare) and ImageQuant LAS4000 (GE Healthcare) were used to detect protein bands. Densitometry analysis was performed using Image J software (NIH, Bethesda, MD, USA).
9
Quantitative Immuno-Dot Blot Analysis
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Immuno-dot and immuno-slotblot assays were performed as previously described61 (link). DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen 69504). DNA (300 ng per well, two to three technical replicates per sample) was vacuum-transferred to a nitrocellulose membrane using the Bio-Dot or Bio-Dot-SF apparatus (Bio-Rad, 1706542/5). Membranes were baked at 80 oC in a Bio-Rad’s Gel Dryer model 583, blocked in 5% milk in PBS with 0.1% Tween (PBST), washed three times in PBST, and incubated with 6-4PP or CPD antibodies (see Supplementary Table 5 ) overnight at 4 oC. Membranes were again washed in PBST and incubated with HRP-conjugated anti-mouse antibodies (ECL Mouse IgG, HRP-linked whole Ab (from sheep), Cytiva, NA931). Damage signal was detected using enhanced chemiluminescence (ECL™ Prime Western Blotting System, GE Healthcare, RPN2232) and exposure in the Bio-Rad ChemiDocTM XRS + imaging system. Genomic DNA amount loaded onto the membrane was quantified using SYBR™ Gold Nucleic Acid Gel Stain (1:5000 dilution, Invitrogen, S11494), by incubating the membrane with SYBR-Gold solution for 60 min, followed by three washes with PBST. Damage signal was normalized to SYBR-Gold signal using Image Lab version 6.0 from Bio-Rad.
10
Protein Separation and Detection Protocols
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HeLa cell lysates (15 μg), raw and solubilised E. coli membranes, affinity and size exclusion chromatography fractions, or purified pore proteins (2.2 ng) were separated on NuPAGE Novex 4–12% BisTris SDS gels (Life Technologies) and where indicated, stained with Instant Blue coomassie stain (Expedeon).
For Western blotting, proteins were transferred to PVDF (BioRad or iBlot, Life Technologies) membranes according to standard procedures. The primary antibodies used were anti-GFP (rabbit polyclonal α-GFP, A11122 Life Technologies, 1 in 1000) and anti-TPC2 (rabbit polyclonal α-TPC2, Eurogentec custom antibody, 1 in 1000)24 (link). Blots were developed using a secondary antibody (goat α-rabbit IgG/horseradish peroxidase (HRP) conjugate, 1706515 BioRad, 1 in 2000) and the ECL Prime Western Blotting System (GE Healthcare). All antibodies were incubated for 1 hr at room temperature. His-tagged proteins were detected using a monoclonal α-poly-histidine/alkaline phosphatase conjugate antibody (mouse, A5588 Sigma, 1 in 2000, 2 hrs at room temperature). Blots were developed using SIGMAFAST BCIP/NBT tablets (Sigma), as per the manufacturer’s instructions.
For Western blotting, proteins were transferred to PVDF (BioRad or iBlot, Life Technologies) membranes according to standard procedures. The primary antibodies used were anti-GFP (rabbit polyclonal α-GFP, A11122 Life Technologies, 1 in 1000) and anti-TPC2 (rabbit polyclonal α-TPC2, Eurogentec custom antibody, 1 in 1000)24 (link). Blots were developed using a secondary antibody (goat α-rabbit IgG/horseradish peroxidase (HRP) conjugate, 1706515 BioRad, 1 in 2000) and the ECL Prime Western Blotting System (GE Healthcare). All antibodies were incubated for 1 hr at room temperature. His-tagged proteins were detected using a monoclonal α-poly-histidine/alkaline phosphatase conjugate antibody (mouse, A5588 Sigma, 1 in 2000, 2 hrs at room temperature). Blots were developed using SIGMAFAST BCIP/NBT tablets (Sigma), as per the manufacturer’s instructions.
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