5-IAF (5-Iodoacetamidofluorescein)
(Synonyms: 5-碘乙酰氨基荧光素; 5-Iodoacetamidofluorescein) 目录号 : GC34180
A fluorescent probe for labeling proteins
Cas No.:63368-54-7
Sample solution is provided at 25 µL, 10mM.
;)
,-1-7$$$37316672.jpg');)
;)
;)
$$$36806353.jpg');)
;33(7)%EF%BC%9A546-561$$$37156877.jpg');)
;)
;)
;)
%201124-1138.$$$35908550.jpg');)
%20766-780.$$$37100057.jpg');)
%20418-432.$$$36893736.jpg');)
%EF%BC%9A-240-255.$$$35108512.jpg');)
533-545$$$36543525.jpg');)
%201-13.$$$36600053.jpg');)
;)
Quality Control & SDS
- View current batch:
- Purity: >95.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
|
Kinase experiment: |
HepG2 cells are lysed in 1 mL 2% acetonitrile in water, and transfered to a 1.5 mL centrifuge tube. An additional 200 µL 2% acetonitrile is used to rinse the dish and combined with the cell extract. To measure the free GSH concentration, three aliquots of 80 µL cell extract are immediately transferred to 0.5 mL centrifuge tubes after a quick mix. Then 20 µL 50 µM NAC is added, followed by the addition of 100 µL 400 µM 5-IAF to the mixture. The derivatization is conducted at room temperature in dark for 1 h, after which the mixtures are centrifuged at 9000× g for 5 min to sediment the insoluble proteins. 10 µL supernatant is diluted 100× with water and analyzed immediately by CE-LIF[2]. |
|
References: [1]. Salvatore Sotgia, et al. Plasma L-Ergothioneine Measurement by High-Performance Liquid Chromatography and Capillary Electrophoresis after a Pre-Column Derivatization with 5-Iodoacetamidofluorescein (5-IAF) and Fluorescence Detection. PLoS One. 2013; 8(7): e70374. |
|
5-IAF is a fluorescent probe for labeling proteins.1 It has been used to label proteins through a reaction with sulfhydryl groups. 5-IAF displays excitation/emission maxima of 491/518 nm, respectively, which can shift during protein labeling or changes in hydrophobicity and can be quenched by potassium iodide. 5-IAF has been used to monitor ligand binding and conformational changes of the Na+/K+-ATPase.2
1.Hartig, P.R., Bertrand, N.J., and Sauer, K.5-Iodoacetamidofluorescein-labeled chloroplast coupling factor 1: Conformational dynamics and labeling-site characterizationBiochemistry16(19)4275-4282(1977) 2.Kapakos, J.G., and Steinberg, M.Ligand binding to (Na,K)-ATPase labeled with 5-iodoacetamidofluoresceinJ. Biol. Chem.261(5)2084-2089(1986)
| Cas No. | 63368-54-7 | SDF | |
| 别名 | 5-碘乙酰氨基荧光素; 5-Iodoacetamidofluorescein | ||
| Canonical SMILES | O=C(NC1=CC2=C(C3(C4=C(OC5=C3C=CC(O)=C5)C=C(O)C=C4)OC2=O)C=C1)CI | ||
| 分子式 | C22H14INO6 | 分子量 | 515.25 |
| 溶解度 | DMF: 30 mg/ml,DMSO: 30 mg/ml,DMSO:PBS (pH 7.2) (1:3): 0.25 mg/ml,Ethanol: 500µ g/ml | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
||
| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
 |
1 mg | 5 mg | 10 mg |
| 1 mM | 1.9408 mL | 9.704 mL | 19.4081 mL |
| 5 mM | 0.3882 mL | 1.9408 mL | 3.8816 mL |
| 10 mM | 0.1941 mL | 0.9704 mL | 1.9408 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
Plasma L-ergothioneine measurement by high-performance liquid chromatography and capillary electrophoresis after a pre-column derivatization with 5-Iodoacetamidofluorescein (5-IAF) and fluorescence detection
PLoS One 2013 Jul 29;8(7):e70374.PMID:23922985DOI:10.1371/journal.pone.0070374.
Two sensitive and reproducible capillary electrophoresis and high-performance liquid chromatography-fluorescence procedures were established for quantitative determination of L-egothioneine in plasma. After derivatization of L-ergothioneine with 5-Iodoacetamidofluorescein, the separation was carried out by HPLC on an ODS-2 C-18 sperisorb column by using a linear gradient elution and by HPCE on an uncoated fused silica capillary, 50 µm id, and 60 cm length. The methods were validated and found to be linear in the range of 0.3 to 10 µmol/l. The limit of quantification was 0.27 µmol/l for HPCE and 0.15 µmol/l for HPLC. The variations for intra- and inter-assay precision were around 6 RSD%, and the mean recovery accuracy close to 100% (96.11%).
Identification of the 5-Iodoacetamidofluorescein reporter site on the Na,K-ATPase
J Biol Chem 1989 Jan 15;264(2):726-34.PMID:2536022doi
5-Iodoacetamidofluorescein (5-IAF) labels the catalytic (alpha) subunit of dog kidney Na,K-ATPase without inhibiting enzymatic activity and is thus a useful fluorescent reporter of enzyme conformation under conditions of enzyme turnover. In this study conditions for labeling a unique sulfhydryl group are described, and this residue is identified in the cDNA-derived sequence. Reaction with iodoacetate (IAA) prior to fluorescent labeling lowers the stoichiometry of 5-IAF incorporation from 2.1 to 1.2 mol/mol alpha beta protomer, and increases the conformationally dependent fluorescence changes by 40-50%, consistent with the elimination of nonspecific labeling. IAA/IAF-enzyme has the same catalytic activity as the IAF-enzyme. In contrast, treatment with iodoacetamide prior to labeling with 5-IAF abolishes all fluorescence responses, although activity is retained. IAA/IAF-enzyme was digested by extensive trypsinolysis, and the fluorescent peptides released from the membrane were purified by high performance liquid chromatography and sequenced. Several fluorescent peptides were found, containing all or part of the sequence Cys-Ile-Glu-Leu-Cys-Cys-Gly-Ser-Val-Lys, corresponding to residues 452-461 in the sheep alpha subunit. The major site of modification is the second of the vicinal cysteine residues, Cys-457. Phenylarsine oxide, a reagent specific for vicinal sulfhydryl groups, prevents fluorophore incorporation, thereby confirming the identification of the IAF site from the sequence data.
5-Iodoacetamidofluorescein-labeled (Na,K)-ATPase. Steady-state fluorescence during turnover
J Biol Chem 1986 Feb 15;261(5):2090-5.PMID:3003094doi
The fluorescence of (Na,K)-ATPase labeled with 5-Iodoacetamidofluorescein was studied under turnover conditions. At 4 degrees C the hydrolysis of ATP is slowed sufficiently to permit study of the effects of Na+, K+, and ATP on the steady-state intermediates. With Na+ and Mg2+ (Na-ATPase conditions), addition of ATP produces a 7% drop in signal that reverts back to the initial, high fluorescence after a steady state of several minutes. K-sensitive phosphoenzyme is formed under these conditions, indicating that the fluorescence signal during the steady state is associated with E2P. Under (Na,K)-ATPase conditions (Na+, K+, Mg2+), micromolar ATP produces a steady-state signal that is 25% lower than the initial fluorescence, with no detectable phosphoenzyme formed. This low-fluorescence intermediate, which is also formed by adding K+ to enzyme in the Na-ATPase steady state described above, resembles the state produced by adding K+ directly to enzyme under equilibrium conditions, i.e. E2K. The K0.5(K+) for the fluorescence decrease and for keeping the enzyme dephosphorylated are nearly identical, indicating that the fluorescence change accompanies K+-dependent dephosphorylation. High ATP increases the steady-state fluorescence during the (Na,K)-ATPase reaction; while oligomycin produces still another steady-state fluorescent intermediate. These last two intermediates may be associated with the formation of E2P and E1P, respectively.
Studies on conformational changes in Na,K-ATPase labeled with 5-Iodoacetamidofluorescein
J Biol Chem 1989 Feb 15;264(5):2726-34.PMID:2536722doi
The rates of individual steps in the reaction cycle of dog kidney Na,K-ATPase labeled with iodoacetamidofluorescein (IAF) were measured using the fluorescence stopped-flow technique. The maximal rate of the fluorescence quenching accompanying ATP hydrolysis at 20 degrees C in the presence of K+ is 66.3 s-1, while the turnover rate in the same conditions is 15.5 s-1. The rate without K+ is slightly lower. Unexpectedly, at very high ionic strength, K+ accelerates the rate 2-fold. The fluorescence change appears to be associated with the E1P----E2P transition. The results are consistent with the classical Albers-Post scheme but do not support recent criticisms that E1P is kinetically incompetent in the presence of Na+ plus K+. As expected, in the absence of ATP the rate of E2(K)----E1Na was very slow (0.2 s-1) but was greatly accelerated by ATP (maximal rate 15.9 s-1) with low affinity (K0.5 = 196 microM). It was concluded that E2(K)----E1 is the slowest step of the cycle, even at nonlimiting ATP concentrations. The rate of E1K----E2(K) for both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme was stimulated by K+ acting with low affinity, but not at all by ATP at 5 microM. Whereas the maximal rate with IAF-enzyme (271 s-1) was similar to previous work, the K+ affinity was significantly higher. Fluorescence signals accompanying hydrolysis of acetyl phosphate with both IAF- and fluorescein 5'-isothiocyanate-labeled enzyme have similar rates, 5.25 s-1 and 4.06 s-1, respectively. A species difference was observed between dog and pig kidney Na,K-ATPase in that both enzymes are labeled with IAF but only in dog enzyme were conformational transitions associated with fluorescence changes. Therefore, the IAF-labeled dog kidney enzyme is the preparation of choice for measuring fluorescence changes accompanying ATP hydrolysis.
Ligand binding to (Na,K)-ATPase labeled with 5-Iodoacetamidofluorescein
J Biol Chem 1986 Feb 15;261(5):2084-9.PMID:3003093doi
The equilibrium binding of sodium, potassium, and adenine nucleotides to dog kidney (Na,K)-ATPase was studied by measuring changes in the fluorescence of enzyme labeled with 5-Iodoacetamidofluorescein (5-IAF). The intensity of the fluorescence emission at 520 nm of the bound fluorescein (excited at 490 nm) is increased by ATP, adenyl-5'-yl imidodiphosphate (AMP-PNP), ADP (but not AMP), and Na+, and decreased by K+, Rb+, NH+4, and LI+. Thus the fluorescence effects correlate with the ability of these groups of ligands to stabilize E1 and E2 conformations, respectively. The Na+-induced increase in fluorescence has two components: a slow, high-affinity increase of approximately 7% (K0.5 = 0.16 mM) with positive cooperativity; and a large (approximately 15%), rapid, low-affinity (K0.5 = 34 mM) increase that is not cooperative. The K0.5 for the high-affinity effect is decreased by oligomycin and increased by K+. ATP effects on the fluorescence follow Michaelis-Menten kinetics and are of high affinity (K0.5 = 0.12 microM); K+ increases the K0.5 for ATP, AMP-PNP, and ADP but does not induce cooperative behavior. K+ itself decreases the fluorescence signal by about 9%, with high affinity (K0.5 = 5 microM), showing Michaelis-menten behavior in the absence of other ligands, while with ATP, Na+, or Mg2+ present, K+ effects are cooperative and of lower affinity.