5-IAF (Synonyms: 5-Iodoacetamidofluorescein)

Name: 5-IAF
Brand: MedChemExpress
SKU: HY-D0807
Rating: 4.5 (1 reviews)

Cat. No.: HY-D0807 Purity: ≥95.0%: FAQs
Data Sheet SDS COA Handling Instructions

Molarity Calculator

5-IAF (5-Iodoacetamidofluorescein) is an idoacetamide derivate of fluoresceine. 5-IAF can be used as fluorescent probe that labels proteins and other molecules having free thiols (cysteine side chains).

For research use only. We do not sell to patients.

5-IAF Chemical Structure

CAS No. : 63368-54-7

Size	Price	Stock	Quantity
5 mg	USD 84	In-stock	Estimated Time of Arrival: December 31
10 mg	USD 134	In-stock	Estimated Time of Arrival: December 31
50 mg		Get quote
100 mg		Get quote

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Based on 1 publication(s) in Google Scholar

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Description

In Vitro

8 μM GSH sample solution is derivatized with 8, 80, 200, 400, 800, 1600, 2000, 2500 μM 5-IAF, respectively and analyzed with CE-LIF. The highest signal intensity is obtained with 400 and 800 μM 5-IAF^[2].

MedChemExpress (MCE) has not independently confirmed the accuracy of these methods. They are for reference only.

Molecular Weight

515.25

Formula

C₂₂H₁₄INO₆

CAS No.

63368-54-7

Appearance

Solid

Color

Light yellow to orange

Emission (Em)

515

Excitation (Ex)

488

SMILES

O=C(NC1=CC2=C(C3(C4=C(OC5=C3C=CC(O)=C5)C=C(O)C=C4)OC2=O)C=C1)CI

Shipping

Room temperature in continental US; may vary elsewhere.

Storage

-20°C, protect from light

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

Solvent & Solubility

In Vitro:

DMSO : 50 mg/mL (97.04 mM; Need ultrasonic; Hygroscopic DMSO has a significant impact on the solubility of product, please use newly opened DMSO)

Preparing
Stock Solutions

Concentration Solvent Mass	1 mg	5 mg	10 mg

1 mM	1.9408 mL	9.7040 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

View the Complete Stock Solution Preparation Table

* Please refer to the solubility information to select the appropriate solvent. Once prepared, please aliquot and store the solution to prevent product inactivation from repeated freeze-thaw cycles.
Storage method and period of stock solution: -80°C, 6 months; -20°C, 1 month (protect from light). When stored at -80°C, please use it within 6 months. When stored at -20°C, please use it within 1 month.

Molarity Calculator
Dilution Calculator

Mass (g) = Concentration (mol/L) × Volume (L) × Molecular Weight (g/mol)

Concentration _(start) × Volume _(start) = Concentration _(final) × Volume _(final)

This equation is commonly abbreviated as: C₁V₁ = C₂V₂

Concentration _(start)

Volume _(start)

Concentration _(final)

Volume _(final)

In Vivo:

Select the appropriate dissolution method based on your experimental animal and administration route.

For the following dissolution methods, please ensure to first prepare a clear stock solution using an In Vitro approach and then sequentially add co-solvents:
To ensure reliable experimental results, the clarified stock solution can be appropriately stored based on storage conditions. As for the working solution for in vivo experiments, it is recommended to prepare freshly and use it on the same day.
The percentages shown for the solvents indicate their volumetric ratio in the final prepared solution. If precipitation or phase separation occurs during preparation, heat and/or sonication can be used to aid dissolution.

Protocol 1

Add each solvent one by one: 10% DMF 40% PEG300 5% Tween-80 45% Saline
Solubility: ≥ 2.5 mg/mL (4.85 mM); Clear solution
Protocol 2

Add each solvent one by one: 10% DMF 90% (20% SBE-β-CD in Saline)
Solubility: 2.5 mg/mL (4.85 mM); Suspended solution; Need ultrasonic
Protocol 3

Add each solvent one by one: 10% DMSO 40% PEG300 5% Tween-80 45% Saline
Solubility: 1.43 mg/mL (2.78 mM); Suspended solution; Need ultrasonic

This protocol yields a suspended solution of 1.43 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (14.3 mg/mL) to 400 μL PEG300, and mix evenly; then add 50 μL Tween-80 and mix evenly; then add 450 μL Saline to adjust the volume to 1 mL.
Preparation of Saline: Dissolve 0.9 g sodium chloride in ddH₂O and dilute to 100 mL to obtain a clear Saline solution.
Protocol 4

Add each solvent one by one: 10% DMSO 90% (20% SBE-β-CD in Saline)
Solubility: 1.43 mg/mL (2.78 mM); Suspended solution; Need ultrasonic

This protocol yields a suspended solution of 1.43 mg/mL. Suspended solution can be used for oral and intraperitoneal injection.
Taking 1 mL working solution as an example, add 100 μL DMSO stock solution (14.3 mg/mL) to 900 μL 20% SBE-β-CD in Saline, and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C, storage for one week): 2 g SBE-β-CD powder is dissolved in 10 mL Saline, completely dissolve until clear.

In Vivo Dissolution Calculator

Please enter the basic information of animal experiments:

Dosage

mg/kg

Animal weight
(per animal)

Dosing volume
(per animal)

μL

Number of animals

Recommended: Prepare an additional quantity of animals to account for potential losses during experiments.

Please enter your animal formula composition:

DMSO +

Tween-80 +

Saline

Recommended: Keep the proportion of DMSO in working solution below 2% if your animal is weak.

The co-solvents required include: DMSO, . All of co-solvents are available by MedChemExpress (MCE). , Tween 80. All of co-solvents are available by MedChemExpress (MCE).

Calculation results:

Working solution concentration: mg/mL

Method for preparing stock solution: mg drug dissolved in μL DMSO (Stock solution concentration: mg/mL).

*In solvent : -80°C, 6 months; -20°C, 1 month (protect from light)

The concentration of the stock solution you require exceeds the measured solubility. The following solution is for reference only. If necessary, please contact MedChemExpress (MCE).

Method for preparing in vivo working solution for animal experiments: Take μL DMSO stock solution, add μL . μL , mix evenly, next add μL Tween 80, mix evenly, then add μL Saline.

If the continuous dosing period exceeds half a month, please choose this protocol carefully.

Please ensure that the stock solution in the first step is dissolved to a clear state, and add co-solvents in sequence. You can use ultrasonic heating (ultrasonic cleaner, recommended frequency 20-40 kHz), vortexing, etc. to assist dissolution.

Purity & Documentation

Purity: ≥95.0%

Select Batch:

Data Sheet (279 KB) SDS (252 KB)

COA (257 KB)

Handling Instructions (2659 KB)

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. [Content Brief]

[2]. Yan Wang, et al. Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A. 2009 Apr 17;1216(16):3533-7. [Content Brief]

Kinase Assay
^[2]

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].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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. [Content Brief]

[2]. Yan Wang, et al. Determination of free and protein-bound glutathione in HepG2 cells using capillary electrophoresis with laser-induced fluorescence detection. J Chromatogr A. 2009 Apr 17;1216(16):3533-7. [Content Brief]

Complete Stock Solution Preparation Table

Optional Solvent	Concentration Solvent Mass	1 mg	5 mg	10 mg	25 mg

DMSO	1 mM	1.9408 mL	9.7040 mL	19.4081 mL	48.5201 mL
	5 mM	0.3882 mL	1.9408 mL	3.8816 mL	9.7040 mL
	10 mM	0.1941 mL	0.9704 mL	1.9408 mL	4.8520 mL
	15 mM	0.1294 mL	0.6469 mL	1.2939 mL	3.2347 mL
	20 mM	0.0970 mL	0.4852 mL	0.9704 mL	2.4260 mL
	25 mM	0.0776 mL	0.3882 mL	0.7763 mL	1.9408 mL
	30 mM	0.0647 mL	0.3235 mL	0.6469 mL	1.6173 mL
	40 mM	0.0485 mL	0.2426 mL	0.4852 mL	1.2130 mL
	50 mM	0.0388 mL	0.1941 mL	0.3882 mL	0.9704 mL
	60 mM	0.0323 mL	0.1617 mL	0.3235 mL	0.8087 mL
	80 mM	0.0243 mL	0.1213 mL	0.2426 mL	0.6065 mL

5-IAF Related Classifications

Help & FAQs

Do most proteins show cross-species activity?
Species cross-reactivity must be investigated individually for each product. Many human cytokines will produce a nice response in mouse cell lines, and many mouse proteins will show activity on human cells. Other proteins may have a lower specific activity when used in the opposite species.

Keywords:

5-IAF63368-54-75-IodoacetamidofluoresceinFluorescent Dyefluorescent probecysteineprotein labelingInhibitorinhibitorinhibit

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5-IAF (Synonyms: 5-Iodoacetamidofluorescein)

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