Total cell lysates were electrophoresed on 5–20% SDS-polyacrylamide gels, and the fractionated products were electroblotted onto Hybond-P membranes (GE Healthcare Bio-Sci. Corp., Piscataway, NJ). The membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) or ECL Prime Blocking reagent (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) for 30 min at RT55 (link),56 (link). The following antibodies were then applied: mouse anti-XLF monoclonal antibody (D-1, 1:500) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-GFP polyclonal antibody (FL, 1:4000) (Santa Cruz Biotechnology), or mouse β-actin monoclonal antibody (AC-15, 1:500) (Sigma-Aldrich). The antibodies were diluted in Signal Enhancer HIKARI (Nacalai Tesque). Binding to each protein was detected using the Select Western blotting detection system (GE Healthcare Bio-Sciences) and visualized using the ChemiDoc XRS system (Bio-Rad, Hercules, CA, USA).
Ecl prime blocking reagent
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
ECL Prime Blocking Reagent is a laboratory reagent used to block non-specific binding in Western blotting and other immunoassay techniques. It is designed to reduce background signal and improve the specificity of target protein detection.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs system, by Bio-Rad (2 mentions)
Hybond, by Cytiva (2 mentions)
Phosstop, by Roche (2 mentions)
Las 4000, by Cytiva (2 mentions)
Polyvinylidene difluoride membrane, by Merck Group (2 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (2 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ecl prime western blotting detection kit, by GE Healthcare (2 mentions)
Signal enhancer hikari, by Nacalai Tesque (1 mentions)
Rabbit anti gfp polyclonal antibody fl, by Santa Cruz Biotechnology (1 mentions)
Blocking one, by Nacalai Tesque (1 mentions)
Complete mini, by Roche (1 mentions)
Ecl prime detection reagent, by GE Healthcare (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Hrp conjugated anti rabbit immunoglobulin, by Cell Signaling Technology (1 mentions)
Can get signal, by Toyobo (1 mentions)
Lysopure nuclear and cytoplasmic extractor kit, by Fujifilm (1 mentions)
Trans blot, by Bio-Rad (1 mentions)
Supersignal west femto maximum sensitivity substrate, by Thermo Fisher Scientific (1 mentions)
16 protocols using ecl prime blocking reagent
1
Western Blot Analysis of Protein Lysates
2
Western Blotting of MAPK Activation
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Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with inhibitors of proteinases and phosphatases (Complete Mini and PhosSTOP; Roche Diagnostics, Rotkreuz, Switzerland). The cell lysate with 20 μg of protein was electrophoresed in a 10–20% gradient polyacrylamide gel (DRC, Tokyo, Japan) and transferred to Clear Blot Membrane-p (ATTO, Tokyo, Japan). The blot was blocked using PBS with 0.1% Tween-20 containing 2% bovine serum albumin or 0.8% ECL Prime Blocking Reagent (GE Healthcare, Chicago, IL, USA). Primary antibodies employed were monoclonal anti-MAP kinase, activated (diphosphorylated ERK1 and 2) (clone MAPK-YT; 1:3000; Sigma-Aldrich), a monoclonal anti-p44/42 MAPK (Erk1/2) (clone 137F5; 1:1500; Cell Signaling Technology, Danvers, MA, USA), and monoclonal anti-beta actin (clone AC-15; 1:2000; Sigma-Aldrich). The secondary antibodies employed were a horseradish peroxidase (HRP)-conjugated anti-mouse immunoglobulin (1:30,000; GE Healthcare) or an HRP-conjugated anti-rabbit immunoglobulin (1:10,000; Cell Signaling Technology). These antibodies were diluted with Can Get Signal (TOYOBO, Osaka, Japan) according to the manufacturer’s instructions. Signals were visualized by the reaction with ECL Prime Detection Reagent (GE Healthcare) and digitally processed using an LAS 4000 mini-CCD camera system (Fuji Photo Film, Tokyo, Japan).
3
Western Blot Analysis of Parasite-Infected Cells
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HFF cells were infected with parasites (multiplicity of infection = 3) for 24 h, then lysed using the LysoPure™ Nuclear and Cytoplasmic Extractor Kit (Wako, Osaka, Japan) supplemented with complete mini protease inhibitors and Phos stop (Roche, Mannheim, Germany). The cell lysates were separated by SDS-polyacrylamide gel electrophoresis and transferred to a Poly Vinylidene Di-Fluoride membrane (Millipore), which was blocked in TBS/0.1% Tween-20/2% ECL Prime Blocking Reagent (GE Healthcare, Buckinghamshire, UK) and incubated with primary and secondary antibodies. The protein bands were visualized by ECL Prime Western Blotting Detection reagent (GE Healthcare), and analyzed by Versa Doc with Quantity One (Bio-Rad, Munich, Germany). Band intensity was quantified using ImageJ software developed by the US National Institutes of Health.
4
Western Blot Protein Separation and Detection
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Ten Vl whole cell extracts of 2 x 105 cells in 40 μl 6X SDS loading buffer were run on 4–14% bis-tris gel (Invitrogen cat # NP0335). Membranes were transferred by semi-dry apparatus (Bio-Rad Transblot cat # 170–3940) at 200 mA, 25 V for 35 min to 0.45 μm nitrocellulose membrane (Millipore cat # IPVH00010). Membranes were then blocked for 1 hr with TBS-T 1% ECL Prime blocking reagent (GE Healthcare cat # RPN418) at 25°C on an orbital shaker and blotted with primary antibody for 1 hr at 25°C with gentle agitation. Membranes were then washed three times for 5 min while shaking with TBS-T and then incubated with secondary antibody at 25°C for 1 hr while shaking. A complete list of antibodies can be found in Supplementary file 2 .
5
Protein Extraction and Western Blotting
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Dissected cerebellum was lysed with SDS lysis buffer (1% SDS, 10% glycerol, 5% β-mercaptoethanol and 125 mM Tris-HCl, pH 6.8). The tissue was homogenized and then boiled at 100 °C for 10 min. To extract proteins from HEK293A cells, the cells were lysed with NP-40 lysis buffer (1% NP-40, 0.5% sodium deoxycholate, 150 mM NaCl and 50 mM Tris-HCl, pH 7.5, with Complete protease inhibitor cocktails (Roche)). All lysates were centrifuged and the supernatant were collected for separation by 10% SDS–polyacrylamide gel electrophoresis. The separated proteins were blotted to polyvinylidene difluoride membrane by Bio-Rad Trans-Blot Turbo Transfer System. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 for 1 h at room temperature. The blocked membrane was incubated with 1:1,000 mouse anti-Espin (BD Biosciences, 611656) or 1:1,000 rabbit anti-β-actin (Cell Signaling, 4967S) or 1:1,000 rabbit anti-FLAG (Sigma, F7425) diluted in 2% ECL Prime Blocking Reagent (GE Healthcare) at 4 °C overnight. After washing, the membrane was incubated with respective secondary antibodies conjugated to horseradish peroxidase (HRP) (GE Healthcare) for 1 h at room temperature. The signal was detected using ECL Western Detection Reagent (GE Healthcare) or SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Scientific).
6
Verification of CRT PAb Specificity in Hyacinth
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The specificity of the CRT PAb for hyacinth was verified by Western blot analysis according to a previously described protocol (Lenartowski et al. 2015 (link)). In brief, 100 mg of whole hyacinth and maize (positive control) pistils was powdered in liquid nitrogen, and the soluble proteins were extracted with 50 mM Tris–HCl (pH 7.5), 1 mM EGTA, 2 mM DTT plus 1 mM PMSF, and Complete Protease Inhibitor Cocktail (Roche). After centrifugation at 16,000 g for 30 min at 4 °C, the supernatants were collected, and the protein concentrations were determined by the Lowry method (Bio-Rad DC Protein Assay). Equal amounts of proteins (15 μg/lane) were separated by electrophoresis on a 12.0 % SDS-PAGE gel, transferred to Hybond-P membrane (GE Healthcare), and blocked with 2 % ECL Prime™ blocking reagent (GE Healthcare). The blots were probed with CRT PAb, washed, and then re-probed with anti-rabbit IgG antibody that was conjugated with horseradish peroxidase (HRP, Sigma). The final detection was performed with the Amersham ECL Advance Western Blotting Detection Kit according to the manufacturer’s guidelines (GE Healthcare).
7
Immunoblot Analysis of Protein Samples
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Cells were lysed in a lysis buffer solution (20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% SDS), followed by SDS gel-electrophoresis and semi-dry transfer of the proteins to polyvinylidene difluoride (PVDF) membranes. All sample protein concentrations were measured using the BCA Protein Assay Kit (Thermo Scientific, Waltham, MA), and the same amounts of protein were applied. Non-specific binding of proteins to the membranes was blocked by incubation in TBS-T buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.1% Tween-20) with 2% ECL Prime Blocking Reagent (GE Healthcare, Buckinghamshire, UK), and the membranes were then incubated with primary antibodies. The antibodies and dilutions used in these studies are described below. Immunodetection was performed with the ECL Prime Western Blotting Detection Kit (GE Healthcare). Pictures were taken with a cold CCD camera (EZ-Capture MG; ATTO, Tokyo, Japan).
8
SDS-PAGE and Western Blot Analysis
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40 μg of protein from each extract or subcellular fraction were separated on pre-made Mini Protean TGX gels (Bio-Rad) and run at 180 V for 35 min in 1xTGS buffer (25 mM Tris, 250 mM Glycine, 0.1% SDS). Gels were transferred via a Mini-Transblot Cell (Bio-Rad), at 55 V for 90 min at 4°C onto Immun-Blot PVDF membrane (BioRad) in 1xTransfer buffer (25 mM Tris, 192 mM Glycine, 20% (v/v) Methanol). Membranes were blocked in 2% ECL prime blocking reagent (GE Healthcare) in 1xTBST solution (20 mM Tris and 150 mM NaCl, 0.1% Tween-20). Anti-GAPDH (10494-I-AP, Proteintech), anti-TBP (8515S, Cell Signaling Technology), or anti-α-Tubulin (ab4074, Abcam) primary antibodies were used at 1:10000, 1:2000, and 1:20000 dilutions, respectively. A 1:20000 dilution of anti-rabbit secondary antibody (NA934-1ML, GE Healthcare) in 0.2% ECL Block in 1x TBST was used for all blots. Blots were developed with ECL Prime Western Blotting Detection Reagent (GE Healthcare) for 2 min before imaging on the BioRad Chemidoc MP Imaging System, using the Blots-Chemi setting.
9
Western Blot Analysis of CYP3A Protein Expression
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Proteins extracted from microsomes (15 μg) were boiled in a quarter-volume of sample buffer (1 M Tris-HCl [pH 7.4], 640 mM 2-mercaptoethanol, 0.2% bromophenol blue, 4% sodium dodecyl sulfate [SDS], and 20% glycerol) and separated on 10% SDS polyacrylamide gels. The proteins on the gels were transferred to polyvinylidene difluoride membranes. The membranes were then blocked with 1% ECL Prime Blocking Reagent (GE Healthcare, Buckinghamshire, England) in Tris-buffered saline (TBS) containing 0.5% Tween 20 for 1 h at room temperature. Anti-CYP3A goat polyclonal antibody L-14 (1:350; sc-30621, Santa Cruz Biotechnology, Santa Cruz, CA, USA) and anti-β-actin rabbit monoclonal antibody 13E5 (1:2,000; #4970, Cell Signaling, Beverly, MA, USA) were used as primary antibodies. Membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. The secondary anti-goat IgG antibody (Santa Cruz Biotechnology) and anti-rabbit IgG antibodies (GE Healthcare) were diluted to 1:10,000 Protein/antibody complexes were visualized with Chemi-Lumi One Super (Nacalai Tesque) and detected with an Image Quant LAS 4000 (GE Healthcare). The intensities of the bands were quantified with Image Quant TL software (GE Healthcare).
10
Protein Extraction and Western Blot Analysis
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Fully grown GV-stage oocytes from each group were isolated as mentioned earlier, and briefly washed with L-15 medium. Then, 30 oocytes per tube were collected in 10 µl of L-15 medium and snap-frozen in liquid nitrogen. Frozen oocytes were stored at −80°C before testing. Proteins were extracted using Bio-Rad sample buffer (161-0747) with 2-mercaptoethanol (Sigma-Aldrich; M7522) at 95°C for 10 min and then centrifuged at 16,000 g for 2 min; the supernatant was then collected. Proteins were separated using Bio-Rad 4-15% gradient gels (456-1084) and blotted on a PVDF membrane (GE Healthcare; 10061-494). Blots were blocked with 0.3% ECL Prime Blocking Reagent (GE Healthcare; RPN418) in Tris-buffered saline supplemented with 0.01% Tween (TBSTw) for 1 h. SMC3 (Abcam; ab9263; 1:2000) and γ-tubulin (Abcam; ab11316; 1:1000) primary antibodies were diluted in 0.3% blocking buffer at 4°C overnight. Blots were washed with TBSTw three times for 10 min each and then incubated with anti-rabbit or anti-mouse HRP-conjugated secondary antibodies (GE Healthcare; NA931V and NA934V, respectively; 1:5000). Blots were washed with TBSTw three times each for 10 min and then developed with a ECL Prime Western Blotting Detection Reagent kit (GE Healthcare; RPN2232).
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