All oligonucleotides were purchased from IDT Technologies, Inc. ATP was purchased from Sigma-Aldrich. Single-stranded circular M13mp18 DNA was obtained using standardized procedures as previously described [31 ]. Primed M13mp18 DNA was obtained by mixing 100 nM M13mp18 (7249 bp) ssDNA and 150 nM of 5′-[T]20TGCGCCGCTACAGGGCGCGTAC-3′ primer in a 10 mM Tris (pH 7.8), 1 mM EDTA and 100 mM NaCl buffer, and heating the mixture in a water bath at 85 °C for 3 min and gradually cooled to room temperature. 5-Iodoacetamidofluorescein (5-IAF) was purchased from Thermo Scientific. All DNA-dependent reactions were carried out in a reaction buffer comprised of 20 mM Tris acetate (pH 7.8), 125 mM potassium acetate and 10 mM magnesium acetate [6 (link),32 (link),33 (link)].
5 iodoacetamidofluorescein 5 iaf
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom, United States
5-Iodoacetamidofluorescein (5-IAF) is a fluorescent labeling reagent used in biochemical and molecular biology applications. It is a derivative of fluorescein that can covalently bind to thiol groups in proteins. The iodoacetamide functional group of 5-IAF reacts with cysteine residues, allowing for the fluorescent labeling of proteins.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Sulfo lc spdp, by Thermo Fisher Scientific (1 mentions)
Polyethylene glycol, by Merck Group (1 mentions)
Lactate dehydrogenase ldh, by Merck Group (1 mentions)
β nicotinamide adenine dinucleotide, by Merck Group (1 mentions)
Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions)
Glutaraldehyde, by Merck Group (1 mentions)
Sodium pyruvate, by Merck Group (1 mentions)
Goat anti rabbit igg h l alexa fluor 488, by Abcam (1 mentions)
3 protocols using 5 iodoacetamidofluorescein 5 iaf
1
Preparation of Primed M13mp18 DNA
2
Sensitive Protein Detection Protocol
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The following materials were used in this study: sulfo-LC-SPDP (sulfosuccinimidyl 6-(3′-(2-pyridyldithio)propionamido)hexanoate; ThermoScientific, Waltham, MA, USA, 21650), TCEP-HC (Tris(2-carboxyethyl)phosphine hydrochloride Thermo Scientific, Waltham, MA, USA, 20490), EDTA (ethylenediaminetetraacetic acid; Sigma Aldrich, Burlington, MA, USA, E9884), MicroDeckTM-G-150 surfaces (Bioactive Surfaces, Galapagar, Madrid, Spain), lactate dehydrogenase (LDH; Sigma Aldrich, Burlington, MA, USA, L1006-12.5KU), biotinylated anti-lactate dehydrogenase (anti-LDH) antibodies (IgG polyclonal antibody; Origene, Rockville, MD, USA, AP21329BT-N), poly(ethylene glycol)-(N-hydroxysuccinimide 5-pentanoate) ether 2-(biotinylamino)ethane (NHS–PEG–biotin; Sigma Aldrich, Burlington, MA, USA, 757799-100μγ), streptavidin (Sigma Aldrich, Burlington, MA, USA, S4762-5 mg), DeepTipTM SiN R11 atomic-force microscopy probes (Bioactive Surfaces, Galapagar, Madrid, Spain), Goat Anti-Rabbit IgG H&L Alexa Fluor® 488 (Abcam, Cambridge, UK, ab150077), 5-iodoacetamidofluorescein (5-IAF; Thermo Scientific, Waltham, MA, USA, 62246), sodium pyruvate (P2256 Sigma-Aldrich, Burlington, MA, USA,), β-nicotinamide adenine dinucleotide (NADH; N8129-500 mg), and glutaraldehyde (50 wt. % in H2O, Sigma Aldrich, Burlington, MA, USA, 340855).
3
Conjugation of (Z(HER2:4))2DCS with 5-IAF
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Prior to coupling to 5-iodoacetamidofluorescein (5-IAF) (Thermo Fisher Scientific, Waltham, MA, USA), (ZHER2:4)2DCS was reduced with 1 μM TCEP for 1 h at room temperature. 5-IAF was added to a final concentration of 10 times molar excess over each of three cysteines in the protein. The sample was incubated for 2 h with shaking. After incubation, the sample was dialyzed against PBS overnight. The efficiency of the reaction was confirmed by mass spectrometry analysis. The conjugation product is referred to as (ZHER2:4)2DCS-FITC.
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