Western blotting was performed as described previously49 (link). In brief, cells were harvested and lysed in 1× RIPA buffer (Sigma, St. Louis, MO) and protein was quantified using the Bradford reagent (BioRad, Marnes-la-Coquette, France). Cell lysates were loaded on SDS-polyacrylamide gels and Western blotting was performed using standard protocols with antibodies against ZC3H12A (GeneTex, CA, USA), and β-ACTIN (Sigma). After reaction with secondary antibodies, the antibody-bound proteins were detected using an ECL Western blotting kit (GE Healthcare Life Sciences, PA).
Ecl western blotting kit
Manufactured by GE Healthcare
Sourced in United Kingdom, United States, Sweden, Germany
The ECL Western blotting kit is a laboratory equipment product designed for the detection and analysis of proteins using the Western blotting technique. The kit provides the necessary reagents and components to perform this analytical procedure.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Thermo Fisher Scientific (4 mentions)
Immobilon p transfer membrane, by Merck Group (3 mentions)
Ripa buffer, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Sds loading buffer, by Bio-Rad (3 mentions)
Ab97040, by Abcam (2 mentions)
Ab207494, by Abcam (2 mentions)
Ab205718, by Abcam (2 mentions)
Sab4800032, by Abcam (2 mentions)
Polyacrylamide gel, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (2 mentions)
Anti rabbit igg, by Cell Signaling Technology (2 mentions)
Anti mouse igg, by Cell Signaling Technology (2 mentions)
Phostop, by Roche (2 mentions)
Mouse anti ty1, by Merck Group (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Zc3h12a, by GeneTex (1 mentions)
β actin, by Merck Group (1 mentions)
33 protocols using ecl western blotting kit
1
Western Blotting of ZC3H12A Protein
2
ABCB1 Expression in TNBC Cell Lines
3
Biotinylated DNA Probe Binding Assay
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The DNA fragments corresponding to the 284-bp upstream region of vtrB were amplified by PCR using 5’-biotinylated primers. The PCR products were separated by agarose gel electrophoresis and purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). This product was used for the biotinylated DNA probe. Increased concentrations of His-VtrAN or ZIP-fused VtrAN proteins were incubated with 4 nM biotinylated DNA probe in the reaction buffer (10 mM Tris-HCl at pH 7.5, 100 mM KCl, 1 mM dithiothreitol, 1 mM EDTA, 0.2% Nonidet P-40, 10% glycerol and 100 ng/ml bovine serum albumin) for 30 min at room temperature. Reaction mixtures were then separated by 6% non-denaturing polyacrylamide gel electrophoresis in TGE buffer (25 mM Tris, 190 mM glycine and 1 mM EDTA) containing 100 mM KCl at 4°C. The DNA probe was electroblotted to a positively charged nylon membrane, UV cross-linked, probed with horseradish peroxidase (HRP)-conjugated streptavidin and developed using the ECL Western blotting kit (GE Healthcare, Buckinghamshire, UK) according to the manufacturer's instructions.
4
Immunoblotting Protein Analysis Protocol
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At the end of each experiment, cells were lysed in RIPA buffer (Cell Signaling Cat# 9806S). Complete protease/phosphatase inhibitor cocktail (Cell Signaling Cat# 5872S) was added to lysis buffer before use. Protein concentration was determined by Bio-Rad DC protein assay (Bio-Rad). The lysates were then clarified by centrifugation (15,000 g, 10 min) and the resulting supernatant used for immunoblotting. Whole cell lysates were mixed with Laemmli sample buffer and boiled. Aliquots containing equal amounts of protein (30–40 μg) were subjected to SDS-PAGE. Subsequently, proteins were transferred to nitrocellulose membranes and the membrane was blocked by incubation with 5% milk in TBS-T buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween 20), for 1 hour at room temperature. The membrane was then incubated overnight at 4°C with the indicated primary antibody in 3% BSA in TBS-T buffer, washed three times in the same buffer and incubated for 1.5 hour with HRP-conjugated secondary antibody. The membrane was then washed three times with TBS-T buffer and visualized by enhanced chemiluminescence (ECL) Western blotting kit according to the manufacturer's instructions (GE Lifesciences, Piscataway, NJ).
5
Expression Analysis of PfSWIB Fusion System
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To analyze the expression of the PfSWIB fusion system in integrated parasites lines, total parasite extracts were prepared by treatment with 0.15% saponin and re-suspended in SDS-loading buffer (Bio-Rad), then separated on a 12% SDS-PAGE gel (Bio-Rad) and subjected to western blot analysis. Total proteins from the 3D7 clone were used as controls. An antibody to aldolase (1:1000 dilution; Roche, Indianapolis, USA) was used as a positive control. Rabbit anti-HA (1:1000 dilution; Abcam, Cambridge, UK) was used to identify PfSWIB fusion proteins in different parasite lines. An enhanced chemiluminescence (ECL) western blotting kit (GE Healthcare, Uppsala, Sweden) was used to develop western blots. The theoretical molecular weight of endogenous PfSWIB, HA tag and HA-FKBP-LID tag is 92kD, 3.5kD and 22kD, respectively. The gray-level image analysis procedure was implemented with ImageJ software. Statistical significance was determined (*P < 0.05, **P < 0.01).
6
Synchronized Parasite Sample Preparation for Western Blotting
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Sample preparation for western blotting was performed as previously described (17 (link)). Briefly, parasites were synchronized at the ring stage, and 200 μL of erythrocytes were collected at different stages of the next generation. Parasites were released from erythrocytes with 0.15% saponin, and boiled in Laemmli buffer at 100°C for 5 min. Extracts were separated on 6%, 8%, or 10% SDS-polyacrylamide gels, and then electroblotted onto Immobilon-P transfer membranes (Millipore). Probing the membranes with specific antibodies followed the standard protocol. Primary antibodies for western blotting included mouse anti-ty1 (used at 1:1000; Sigma, SAB4800032), rabbit anti-aldolase (1:2000; Abcam, ab207494), and rabbit anti-HP1 (1:2000; a gift from Artur Scherf, Institut Pasteur) (38 (link)). Horseradish peroxidase-conjugated secondary antibodies, namely goat anti-mouse IgG (Abcam, ab97040) and goat anti-rabbit IgG (Abcam, ab205718), were applied at 1:5000. Blot signals were detected using the ECL western blotting kit (GE healthcare).
7
Quantification of MMP-8 in Mouth Rinse Samples
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The molecular forms of MMP-8 were detected from mouth rinse samples by a modified enhanced chemiluminescence (ECL) Western blotting kit according to protocols recommended by the manufacturer (GE Healthcare, Amersham, UK) as described earlier by Rautava et al. [41 (link)]. Briefly, the proteins of mouth rinse samples were first separated by electrophoresis and then electro-transferred onto nitrocellulose membranes Protran (Whatman GmbH, Dassel, Germany). The membranes were incubated overnight with monoclonal primary antibodies anti-MMP-8 [42 (link)] and then with horseradish peroxidase-linked secondary antibody (GE Healthcare, Buckinghamshire, UK) for 1 h. The membranes were washed 4 times in TBST between each step for 15 min. The proteins were visualized using the ECL system according to protocol. The recombinant human MMP-8 (100 ng, Calbiochem, Darnstadt, Germany) was used as a positive control.
8
Western Blot Analysis of Malaria Parasites
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Synchronized schizont-stage parasites were isolated by treatment of infected RBC (iRBCs) with 0.15% saponin in PBS on ice for 10 min. Parasite pellets were washed by 1× PBS twice, then dissolved in 100 μl 1× SDS-loading buffer (Bio-Rad) and boiled at 100°C for 5 min. Total proteins were loaded onto SDS-PAGE gels with suitable amount according to the theoretical molecular weight of target proteins. Proteins were transferred to a PVDF membrane by semi-dry (Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell) for 2 h at room temperature or wet transfer (Trans-Blot® Cell) overnight at 4°C, and visualized by exposing with the Bio-Rad ChemiDoc XRS system. The antibody used in this study was mouse anti-GFP (Sigma), anti-HA (Roche) and rabbit anti-aldolase (Abcam). The experiment was done with ECL western blotting kit (GE healthcare).
9
Western Blot Analysis of Signaling Proteins
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The samples used for western blotting were separated by SDS-PAGE. After electrotransfer, the polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany) were probed with anti-GEF-H1, p(Thr18/Ser19)-MLC, MLC, anti-GST (Cell Signaling Technology, Inc., Danvers, MA, USA), anti-LARG, anti-Ect2, anti-RhoA, anti-β-actin, anti-α-tubulin, or anti-FLAG (Sigma-Aldrich, St. Louis, MO, USA) antibodies, followed by horseradish-peroxidase-conjugated goat anti-rabbit or rabbit anti-mouse antibodies (Zymed Laboratories, Inc., South San Francisco, CA, USA). The blots were developed using an ECL Western Blotting Kit (GE Healthcare, Little Chalfont, UK).
10
T Cell Activation and ERK Signaling
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Total T cells were purified by negative selection using BioMag Goat anti-mouse IgG and anti-rat IgG (QIAGEN GmbH, Hilden, German) after 2.4G2 treatment, and incubated with a biotin-conjugated anti-CD3 mAb (2C11, 10 μg/ml, BioLegend, San Diego, USA) for 30 min on ice. Cells were then incubated with streptavidin (20 μg/ml; Sigma-Aldrich Co., St. Louis, USA) for the indicated periods of time at 37°C. For Western blotting, cells were lysed in 5 x lysis buffer containing 125mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol, and 0.04% bromophenol blue. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose membranes (ATTO Co., Tokyo, Japan), and probed with anti-ERK1/2 (Cell Signaling Technology, Danvers, USA) or anti-phospho-ERK1/2 (Cell Signaling Technology, Danvers, USA) antibodies followed by an HRP-conjugated anti-rabbit antibody (Bio-Rad Laboratories, Hercules, USA). Signals were detected using an ECL Western blotting kit (GE Healthcare, Buckinghamshire, UK) and Image Quant LAS 4000 (GE Healthcare).
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