Cell protein lysate was obtained using cell extraction buffer (Life Technologies, Carlsbad, CA, USA) followed by incubation for 30 min, at 4 °C, on a shaker. After centrifugation, the supernatant was collected and submitted to protein quantification by a BCA Protein Assay Kit. SDS-Page and Western blots were performed as described using stain-free gels [15 (link)]. Membranes were incubated with mouse anti-GAPDH, mouse anti-ILK [N1C1] (GeneTex, Irvine, USA), mouse anti-β-actin, mouse anti-β1-integrin P5D2, rabbit anti-CTR1 [FL190], goat anti-MRP2 [H17] (Santa Cruz Biotechnology), as well as goat anti-rabbit, donkey anti-goat and anti-mouse IgG kappa binding protein IgG HRP-conjugated (Santa Cruz Biotechnology) diluted in 1% BSA solution. Western blots were quantified via chemiluminescence using a Clarity Western ECL substrate chemiluminescence kit (BioRad Laboratories GmbH, Munich, Germany). Besides the loading control GAPDH, we also used stainfree total protein normalization. Membranes were photographed and analyzed using a ChemiDoc XRS+ imaging acquiring system (BioRad) and Image Lab software v. 6.0 (BioRad).
Clarity western ecl substrate chemiluminescence kit
Manufactured by Bio-Rad
Sourced in Germany
The Clarity Western ECL substrate chemiluminescence kit is a laboratory equipment product designed for the detection of proteins in Western blot analysis. It provides a chemiluminescent substrate that can be used to visualize and quantify target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Chemidoc xrs imaging acquiring system, by Bio-Rad (6 mentions)
Mouse anti gapdh, by GeneTex (5 mentions)
Image lab software v 6, by Bio-Rad (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (2 mentions)
Rabbit anti p erk1 2 thr202 tyr204, by Cell Signaling Technology (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Mouse anti β actin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti erk1 2, by Cell Signaling Technology (1 mentions)
Mouse anti β actin, by R&D Systems (1 mentions)
Molecular imager chemidoc xrs imaging system, by Bio-Rad (1 mentions)
Proteoblock protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Extraction buffer, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Rabbit anti e cadherin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti erk1 2, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti ddr1, by Cell Signaling Technology (1 mentions)
Proteome profiler antibody array kit, by R&D Systems (1 mentions)
Amicon ultra 10k, by Merck Group (1 mentions)
Anti rabbit igg horseradish peroxidase conjugate, by Bio-Rad (1 mentions)
8 protocols using clarity western ecl substrate chemiluminescence kit
1
Protein Quantification and Western Blotting
2
Western Blot Analysis Optimization
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Cells extraction, lysate quantification, SDS-Page and western blots were performed as described using stain-free gels [20 (link)]. Membranes were incubated with rabbit anti-β-catenin, mouse anti-GAPDH (GeneTex), goat anti-pGsk-3β (Ser9), rabbit anti-LaminB1, mouse anti-pCREB (Ser133), mouse anti-pMEK1/2, mouse anti-β-actin, rabbit anti-FAK, rat anti-pFAK (Y397, R&D Systems) rabbit anti-PI3K, goat anti-pPI3K (Tyr 508), goat anti-AKT1, rabbit anti-pAKT1 (Tyr308), rabbit anti-ERK1/2 (Cell Signaling Technology, Frankfurt am Main, Germany), rabbit anti pERK1/2 (Thr202/Tyr204, Cell Signaling Technology) as well as goat anti-rabbit, goat anti-rat, donkey anti-goat and anti-mouse IgG kappa binding protein IgG HRP-conjugated diluted in 1% BSA solution. If not indicated otherwise antibodies were purchased from Santa Cruz Biotechnology. Western blots were quantified via chemiluminescence using a Clarity Western ECL substrate chemiluminescence kit (BioRad). Besides the loading control β-Actin, we used stainfree total protein normalization. Membranes were photographed and quantified using ChemiDoc XRS+ imaging acquiring system (BioRad) and Image Lab software v. 6.0 (BioRad).
3
Western Blot Analysis of BST-2 and Influenza PA
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Infected or transfected cells were lysed in radioimmunoprecipitation (RIPA) buffer (50 mM Tris-Cl pH 7.4, 120 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, 1% sodium dodecyl sulfate) supplemented with 1% ProteoBlock Protease Inhibitor Cocktail (Thermo Scientific)). Lysates were loaded into 10 or 12% polyacrylamide gels for SDS-PAGE, and subsequently transferred onto nitrocellulose membranes. Membranes were blocked with 5% skim milk in 0.1% Tween 20–Tris-buffered saline (TBS) for at least one hour before probing with antibody. Human BST-2 was detected with a rabbit anti-human BST-2 antibody (Santa Cruz Biotechnology), influenza virus PA with a rabbit anti-PA antibody (Thermo Scientific), and β-actin with a mouse anti-β-actin antibody (Santa Cruz Biotechnology). Secondary antibodies (horseradish peroxidase-conjugated goat anti-rabbit IgG and goat anti-mouse IgG antibodies) were purchased from Santa Cruz Biotechnology. Bands were visualized using the Clarity Western ECL Substrate chemiluminescence kit (Bio-Rad) by film exposure. Films were scanned using the Molecular Imager ChemiDoc XRS+ Imaging System (Bio-Rad) and analyzed by densitometry using ImageJ.
4
Western Blot Analysis of E-cadherin
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Cells were washed twice to exclude dead cells. Then, cells were lysed using extraction buffer (Life Technologies, Carlsbad, CA, USA), supplemented with phenylmethanesulfonylfluoride (0.1 mM PMSF) (Life Technologies, Carlsbad, CA, USA) and a protease inhibitor cocktail (1 μg/mL aprotinin, 1 μg/mL leupeptin) (Life Technologies, Carlsbad, CA, USA), according to manufacturer’s instructions. Cells were then centrifuged and supernatant was collected and submitted to protein quantification by Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc, Waltham, MA USA). SDS-Page and Western Blot were performed, as described in [54 (link)]. Membranes were incubated with rabbit anti- E-cadherin (Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-GAPDH (GeneTex, Irvine, CA, USA), and goat anti-rabbit and donkey anti-mouse IgG HRP-conjugated (Santa Cruz Biotechnology, Dallas, TX, USA) diluted in 1% BSA solution. Western Blot was quantified via chemiluminescence using Clarity Western ECL substrate chemiluminescence kit (BioRad). Membranes were photographed and quantified using ChemiDoc XRS+ imaging acquiring system (BioRad) and Image Lab software (BioRad).
5
Western Blot Analysis of Protein Targets
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Cell protein lysate was obtained using cell extraction buffer (Life Technologies, Carlsbad, CA, USA) followed by incubation for 30 min, at 4 °C, on a shaker. After centrifugation, the supernatant was submitted to protein quantification by a BCA Protein Assay Kit. SDS-Page and Western blots were performed as described using stain-free gels [28 (link)]. Membranes were incubated with mouse anti-GAPDH (GeneTex, Irvine, USA), rabbit anti-pERK 1/2, rabbit anti-Trx1 (Cell Signaling Technology, Frankfurt am Main, Germany), rabbit anti-ERK 1/2, rabbit anti-Nrf2, mouse anti-α-Tubulin (Santa Cruz Biotechnology, Heidelberg, Germany) as well as goat anti-rabbit and anti-mouse IgG kappa binding protein IgG HRP-conjugated (Santa Cruz Biotechnology) diluted in 1% BSA solution. Western blots were quantified via chemiluminescence using a Clarity Western ECL substrate chemiluminescence kit (BioRad Laboratories GmbH, Munich, Germany). Besides the loading controls GAPDH and α-tubulin, we also used stainfree total protein normalization. Membranes were photographed and analyzed using a ChemiDoc XRS + imaging acquiring system (BioRad) and Image Lab software v. 6.0 (BioRad).
6
Western Blot Quantification Protocol
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Cells extraction, lysate quantification, SDS-Page and Western blot were performed as described previously using stainfree gels [22 (link)]. Membranes were incubated with mouse anti-GAPDH (GeneTex, Irvine, CA, USA), mouse anti-pEGFR (Tyr1068, 15A2), mouse anti-BCRP (BXP-21), mouse anti-pHSP27 (Ser82, B-3), mouse anti-HSP27 (F-4), mouse anti-integrin β4 (A9), rabbit anti-ERK1/2 (Cell Signaling Technology, Frankfurt am Main, Germany), rabbit anti pERK1/2 (Thr202/Tyr204, Cell Signaling Technology) rabbit anti-DDR1 (Cell Signaling Technology), rabbit anti-pDDR1 (Tyr513, Cell Signaling Technology) as well as goat anti-rabbit and anti-mouse IgG kappa binding protein IgG HRP-conjugated diluted in 1% BSA solution. If not indicated otherwise, antibodies were purchased from Santa Cruz Biotechnology. Western blot was quantified via chemiluminescence using Clarity Western ECL substrate chemiluminescence kit (BioRad). Besides the loading control GAPDH, we used stainfree total protein normalization. Membranes were photographed and quantified using ChemiDoc XRS+ imaging acquiring system (BioRad) and Image Lab software v. 6.0 (BioRad). Representative uncut Western blots are shown in Figure S5 .
7
Proteome Profiling of Cisplatin-Treated Melanoma
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A Proteome Profiler™ Antibody Array kit (R&D Systems GmbH, Wiesbaden-Nordenstadt, Germany) was performed to screen MV3 cells for certain membrane proteins and the impact of cisplatin treatment thereof. Concisely explained, MV3 melanoma cells were washed twice with PBS to exclude dead cells, before the cell lysis buffer provided in the kit was added; cell lysates were prepared following the manufacturer’s instructions. Then, Pierce™ BCA Protein Assay Kit (LifeTechnologies, Thermo Fisher Scientific Inc, Waltham, MA, USA) was used to quantify total protein. Then, the assay was performed following the manufacturer’s instructions. The proteins present in the antibody arrays were quantified via chemiluminescence after membrane treatment with Clarity Western ECL substrate chemiluminescence kit (BioRad Lab GmbH, Munich, Germany). Membranes were photographed and quantified using ChemiDoc XRS+ imaging acquiring system (BioRad), and Image Lab software (BioRad).
8
Extracellular Vesicle Protein Analysis
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The SEC fractions were concentrated with Amicon ® Ultra 10K (Merck) concentrator. The total protein concentration of samples was measured with a DC protein assay (Bio-Rad, Hercules, CA, USA). On each lane of the 12% m/v SDS-PAGE gel, 30 mg of total protein amount from certain fractions was loaded. The proteins were separated at 100V for 90 min. Next, the proteins were blotted onto a PVDF membrane by using a wet transfer system at 100 V for 1 h. The membrane was blocked with TBST buffer with 3% m/v skim milk for 1 h at 4°C. The proteins were detected using the primary rabbit monoclonal antibodies: anti-Hsp70, anti-TSG101, anti-CD63 (Abcam, Trumpington, UK), and the secondary antibodies anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA). The signals were measured in the ChemiDoc Touch Imaging system (Bio-Rad, USA) with a Clarity Western ECL Substrate chemiluminescence kit (Bio-Rad, USA).
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