Fluorescein-5-isothiocyanate (FITC)

Catalog No.S6928 Batch:S692801

Technical Data

Formula	C₂₁H₁₁NO₅S
Molecular Weight	389.38	CAS No.	3326-32-7
Solubility (25°C)*	In vitro	DMSO	78 mg/mL (200.31 mM)
		Ethanol	10 mg/mL (25.68 mM)
		Water	Insoluble
* <1 mg/ml means slightly soluble or insoluble. * Please note that Selleck tests the solubility of all compounds in-house, and the actual solubility may differ slightly from published values. This is normal and is due to slight batch-to-batch variations. * Room temperature shipping (Stability testing shows this product can be shipped without any cooling measures.)

Preparing Stock Solutions

Biological Activity

Description	Fluorescein-5-isothiocyanate (FITC, Fluorescein isothiocyanate isomer I, 5-FITC) is a fluorescent probe capable of being conjugated to tissue and proteins.
In vitro	1.Protein Preparetion 1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL. 2) The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1m sodium bicarbonate shall be used for adjustment. 3) If the protein concentration is lower than 2mg/ml, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL. 4) The protein must be in the buffer without primary amine (such as Tris or glycine) and ammonium ion, otherwise the labeling efficiency will be affected. 2.Dye Preparation Add anhydrous DMSO into the vial of FITC to make a 10 mM stock solution. Mix well by pipetting or vortex. 3.Calculation of dye dosage The amount of FITC required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of FITC to protein is about 10. Example: assuming the required marker protein is 1 mL 2 mg/mL IgG (MW=150,000), use 1 mL DMSO dissolve 1 mg FITC, the required FITC volume is 40 μL. 4.Run conjugation reaction 1) A good volume of freshly prepared 10 mg/mL FITC is slowly added to 0.5 mL protein sample. In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don't mix well to prevent protein samples from denaturation and inactivation. 2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency. 5.Purify the conjugation The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column. 1) Prepare Sephadex G-25 column according to the manufacture instruction. 2) Load the reaction mixture (From ""Run conjugation reaction"") to the top of the Sephadex G-25 column. 3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface. 4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification. Combine the fractions that contain the desired dye-protein conjugate.

Description

Fluorescein-5-isothiocyanate (FITC, Fluorescein isothiocyanate isomer I, 5-FITC) is a fluorescent probe capable of being conjugated to tissue and proteins.

In vitro

1.Protein Preparetion
1) In order to obtain the best labeling effect, please prepare the protein (antibody) concentration as 2 mg/mL.
2) The pH value of protein solution shall be 8.5±0.5. If the pH is lower than 8.0, 1m sodium bicarbonate shall be used for adjustment.
3) If the protein concentration is lower than 2mg/ml, the labeling efficiency will be greatly reduced. In order to obtain the best labeling efficiency, it is recommended that the final protein concentration range is 2-10 mg/mL.
4) The protein must be in the buffer without primary amine (such as Tris or glycine) and ammonium ion, otherwise the labeling efficiency will be affected.
2.Dye Preparation
Add anhydrous DMSO into the vial of FITC to make a 10 mM stock solution. Mix well by pipetting or vortex.
3.Calculation of dye dosage
The amount of FITC required for reaction depends on the amount of protein to be labeled, and the optimal molar ratio of FITC to protein is about 10.
Example: assuming the required marker protein is 1 mL 2 mg/mL IgG (MW=150,000), use 1 mL DMSO dissolve 1 mg FITC, the required FITC volume is 40 μL.
4.Run conjugation reaction
1) A good volume of freshly prepared 10 mg/mL FITC is slowly added to 0.5 mL protein sample. In solution, gently shake to mix, then centrifuge briefly to collect the sample at the bottom of the reaction tube. Don't mix well to prevent protein samples from denaturation and inactivation.
2) The reaction tubules were placed in a dark place and incubated gently at room temperature for 60 minutes at intervals.For 10-15 minutes, gently reverse the reaction tubules several times to fully mix the two reactants and raise the bar efficiency.
5.Purify the conjugation
The following protocol is an example of dye-protein conjugate purification by using a Sephadex G-25 column.
1) Prepare Sephadex G-25 column according to the manufacture instruction.
2) Load the reaction mixture (From ""Run conjugation reaction"") to the top of the Sephadex G-25 column.
3) Add PBS (pH 7.2-7.4) as soon as the sample runs just below the top resin surface.
4) Add more PBS (pH 7.2-7.4) to the desired sample to complete the column purification.
Combine the fractions that contain the desired dye-protein conjugate.

Protocol (from reference)

Selleck's Fluorescein-5-isothiocyanate (FITC) has been cited by 1 publication

Circular RNA circ-MMP11 Contributes to Lapatinib Resistance of Breast Cancer Cells by Regulating the miR-153-3p/ANLN Axis [ Front Oncol, 2021, 11:639961]	PubMed: 34295807

Circular RNA circ-MMP11 Contributes to Lapatinib Resistance of Breast Cancer Cells by Regulating the miR-153-3p/ANLN Axis [ Front Oncol, 2021, 11:639961]

PubMed: 34295807

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SHIPPING AND STORAGE
Selleck products are transported at room temperature. If you receive the product at room temperature, please rest assured, the Selleck Quality Inspection Department has conducted experiments to verify that the normal temperature placement of one month will not affect the biological activity of powder products. After collecting, please store the product according to the requirements described in the datasheet. Most Selleck products are stable under the recommended conditions.

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