Immunoblotting was performed as described (60 (link)). Briefly, cultured cells were lysed using radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Roche). Proteins were resolved with SDS–polyacrylamide gel electrophoresis, and after transfer to a nitrocellulose membrane were blocked in Odyssey blocking buffer (LI-COR). The following primary antibodies were used: human NAF1 (rabbit, ab157106, 1:1000; Abcam), mouse Naf1 (rabbit, Naf1 394, 1:250; Prosci), Myc (mouse, clone 4A6, 1:1000; Millipore), human dyskerin (rabbit, sc-48794, 1:250; Santa Cruz Biotechnology), and green fluorescent protein (mouse, 7.1 and 13.1, 1:1000; Roche) with loading controls actin (mouse, ab8226, 1:2000; Abcam), tubulin (rabbit, ab6046, 1:5000; Abcam), PARP (rabbit, 9542S, 1:1000; Cell Signaling Technology), or GAPDH (mouse, mAbcam9498, 1:1000; Abcam). Secondary antibodies were conjugated to dyes IR680 or IR800 (donkey, 1:10,000; LI-COR), and blots were visualized using an Odyssey scanner (LI-COR), with exception of the mouse Naf1 antibody that was visualized by horseradish peroxidase–linked antibody (rabbit, 7074, 1:10,000; Cell Signaling Technology) and enhanced chemiluminescent substrate (Thermo Scientific). Cell fractionation was performed using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific).
Clone 4a6
Manufactured by Merck Group
Sourced in Hungary
The clone 4A6 is a laboratory equipment product manufactured by Merck Group. It is a research-grade tool designed for specific applications within scientific and industrial settings. The core function of this product is to enable controlled and precise processes, though its intended use should be determined by the customer's specific requirements.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Ab157106, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Enhanced chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Ab6046, by Abcam (1 mentions)
Ir800, by LI COR (1 mentions)
Ir680, by LI COR (1 mentions)
Sc 48794, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase linked antibody, by Cell Signaling Technology (1 mentions)
Odyssey blocking buffer, by LI COR (1 mentions)
Odyssey scanner, by LI COR (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Western lightning, by PerkinElmer (1 mentions)
β actin, by Merck Group (1 mentions)
Pierce magnetic chip kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Enspire, by PerkinElmer (1 mentions)
Dual luciferase reporter assay system, by Promega (1 mentions)
A11010, by Thermo Fisher Scientific (1 mentions)
Zen 2012, by Zeiss (1 mentions)
4 protocols using clone 4a6
1
Immunoblotting Procedure for Protein Expression
2
Immunoblotting of GFP-tagged and Myc-tagged Receptors
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The monoclonal antibodies to GFP (Clontech, Palo Alto, CA, USA; clone JL-8, used at dilution 1/1000) exhibited an exceptionally low background in total cell extracts of untransfected HEK 293 cells. For the analysis of B2R-GFP in total cell extracts, total cell extracts were recovered and analyzed after SDS/PAGE (9% gel) and protein transfer (Bachvarov et al. 2001 (link)) using the anti-GFP antibodies. Myc-tagged B2R constructs were detected in HEK 293a total cell extracts with the anti-myc monoclonal antibodies, clone 4A6 (Millipore, dilution 1:2000). The staining was detected with the appropriate horseradish peroxydase-conjugated secondary antibodies and a luminescent substrate used as directed (Western Lightning, PerkinElmer). Equal track loading was verified by migrating and transferring the same samples separately and immunoblotting for β-actin (monoclonal from Sigma-Aldrich).
3
Dual Luciferase Assay and ChIP Analysis
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A dual luciferase reporter assay system was used to measure the promoter activity according to manufacturer's instruction (Promega Corporation). The Renilla luciferase driven by TK minimal promoter was used as an internal control to normalize the transfection efficiency. Luciferase activity was measured 48 h post-transfection by using an EnSpire® multimode plate reader (Perkin Elmer, Inc.). ChIP assay was carried out according to the manufacturer's instructions (Pierce™ magnetic ChIP kit; Thermo Fisher Scientific, Inc. cat. no. 25157). Briefly, 2×106 cells were transfected with Myc-tag EHF expression construct and cross-linked in 1% formaldehyde at 37°C for 10 min and lysed in SDS lysis buffer (Pierce magnetic ChIP kit; Thermo Fisher Scientific, Inc.) supplemented with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA) for 10 min on ice. The lysate was sonicated and precleared with protein A agarose/salmon sperm DNA prior to immunoprecipitation with 1 µg of anti-Myc Tag antibody (cat. no. 05-724; Clone 4A6; EMD Millipore) at 4°C overnight. Following wash and elution steps, cross-links were reversed at 65°C for 4 h. The DNA in the immunoprecipitated samples was purified by proteinase K digestion followed by DNA purification. An aliquot of purified DNA was analyzed by quantitative PCR with specific primers.
4
Immunostaining of Agroinfiltrated N. benthamiana Protoplasts
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Immunostaining of N. benthamiana protoplasts prepared from agroinfiltrated plants was performed as described previously, using plants at 2 days post-agroinfiltration (83 (link)). The constructs used for agroinfiltration were ER-GFP (49 (link)), 53U-RdRp-Li1-myc (33 (link)), and a control vector, pCAMBIA1301. The primary antibodies used were anti-c-Myc antibody at 1:200 (clone 4A6; EMD Millipore) and anti-dsRNA antibody at 1:100 (clone J2; English and Scientific Consulting Bt., Szirak, Hungary). The secondary antibody was Alexa Fluor 546-conjugated goat anti-rabbit antibody at 1:1,000 (A11010; Thermo Fisher Scientific). DAPI (4′,6-diamidino-2-phenylindole) staining solution (10 μg/ml) was infiltrated into leaves with a needleless syringe 30 min before imaging. Imaging analyses with a confocal laser scanning microscope (LSM710NLO; Carl Zeiss, Jena, Germany) were performed as described previously (84 (link)). sGFP and mCherry were excited with an argon laser at 488 nm and a diode-pumped solid-state laser at 561 nm, and emitted light was captured in windows of 493 and 556 nm and between 573 and 621 nm, respectively. DAPI was excited with 405-nm light, and emitted light was captured in the range between 410 and 528 nm. All fluorescence images were acquired with sequential scanning, and image processing was performed with the Carl Zeiss ZEN 2012 software.
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