FITC标记抗体

INSTRUCTIONS Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax Number Description 46424 46425 FITC, 1 g FITC, 100 mg Chemical name: Fluorescein isothiocyanate Molecular weight: 389.38 Extinction coefficient: 70,000 M-1 cm-1 (in aqueous buffer, pH 9) Ex/Em wavelength: 494/518 nm CAS # 3326-32-7 OO O O O N C S OHO O O OH N C S 46112 TRITC, 10 mg Chemical name: Tetramethylrhodamine-5- (and 6)-isothiocyanate Molecular weight: 478.97 Extinction coefficient: 100,000 M-1 cm-1 (at 544 nm in methanol) Ex/Em wavelength: 541/572 nm CAS #6749-36-6 ON N+ O O N C S ON N+ O O N C S Cl-Cl- Storage: Upon receipt store at -20°C. Store product in the foil pouch with desiccant to protect from light and moisture. Product is shipped at ambient temperature. Introduction FITC and TRITC are among the most simple and commonly used reagents for protein fluorescent labeling. These isothiocyanates react to amino, sulfhydryl, imidazoyl, tyrosyl or carbonyl groups on proteins; however, only the derivatives of primary and secondary amines generally yield stable products. Reactions are most efficient at pH 8-9 and must be performed in an amine-free buffer such as carbonate/bicarbonate. Antibody and other proteins can be effectively labeled with several fluorophore tags per protein molecule when reacted with a 15- to 20-fold molar excess of isothiocyanate-activated fluorophore. Excess nonreacted and hydrolyzed reagent can be removed by dialysis, gel filtration or dye removal resin. 2081.046424 46425 46112 FITC and TRITC User 铅笔 User 铅笔 User 铅笔 User 铅笔 User 铅笔 Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 2 Procedure for Antibody Labeling with FITC Materials Required • Conjugation buffer: 50 mM borate buffer, pH 8.5 (Product No. 28384) • Antibody solution: dissolve ~1 mg of antibody in 0.5 ml of Conjugation Buffer • Device to remove excess dye, such as a Dye Removal Columns (Product No. 22858), Zeba Desalt Spin Column (Product No. 89891) or Slide-A-Lyzer® Dialysis Cassette 10,000 MWCO (Product No. 66380) Procedure 1. Dissolve FITC in DMF at 10 mg/ml. Mix well to completely dissolve the FITC. 2. Add 15- to 20-fold molar excess of FITC to 0.5 ml of antibody solution and immediately mix the reaction. 3. Incubate for 1 hour at room temperature in the dark. 4. Remove excess and hydrolyzed FITC by gel filtration, dialysis or with a Dye Removal Column. Procedure for Antibody Labeling with TRITC This method is adapted from Larsson.1 Materials Required • Conjugation Buffer: 100 mM carbonate/bicarbonate buffer, pH 9.0 • Antibody solution: dialyze antibody into Conjugation Buffer at 6 mg/ml • Device to remove excess dye, such as a Dye Removal Column (Product No. 22858), Zeba Desalt Spin Column (Product No. 89891) or Slide-A-Lyzer Dialysis Cassette 10,000 MWCO (Product No. 66380) Procedure 1. Dissolve TRITC in DMSO at 1 mg/ml. 2. While stirring, slowly add 35 µl of TRITC to 1 ml of the 6 mg/ml antibody solution and mix thoroughly. 2. Incubate for 2 hours at room temperature in the dark. 3. Remove excess and hydrolyzed TRITC by gel filtration, dialysis or with a Dye Removal Column. Additional Information • Visit our web site for a listing of related products (other fluorophores and convenient labeling kits) and technical resources, such as the following Tech Tips: o Tech Tip #43: Protein Stability and Storage o Tech Tip #6: Extinction Coefficients Guide o Tech Tip #31: Calculate Dye:Protein (F/P) Molar Ratios • Fading (photobleaching in tissue sections) sometimes can be reduced by mounting in an alkaline-buffered media (pH 9).1 There are several reagents that may be used with FITC and/or TRITC derivatives to prevent fading, including n-propyl gallate at 0.1-0.25 M dissolved in glycerol.2 For FITC derivatives, o- or p-phenylenediamine added to the mounting buffer from 1 µg/ml to 1 mg/ml in glycerin also may be used.1,2 User 铅笔 User 铅笔 User 铅笔 User 铅笔 User 铅笔 User 铅笔 User 铅笔 User 铅笔 User 铅笔 Pierce Biotechnology PO Box 117 (815) 968-0747 www.thermo.com/pierce 3747 N. Meridian Road Rockford, lL 61105 USA (815) 968-7316 fax 3 Related Products 28384 BupH Borate Buffer Packs, 40 packs 28372 BupH Phosphate Buffered Saline Packs, 40 packs 22858 Fluorescent Dye Removal Columns 66380 Slide-A-Lyzer Dialysis Cassettes, 10,000 MWCO 89889 Zeba Desalt Spin Columns, 2 ml, 5 each, for 200-700 µl samples 89890 Zeba Desalt Spin Columns, 2 ml, 25 each, for 200-700 µl samples 53029 Pierce NHS-Fluorescein Antibody Labeling Kit 53031 Pierce NHS-Rhodamine Antibody Labeling Kit 53027 Pierce FITC Antibody Labeling Kit 46409 NHS-Fluorescein, 1 g 46410 NHS-Fluorescein, 100 mg 46406 NHS-Rhodamine, 25 mg 53024 DyLight 488 Antibody Labeling Kit 53025 DyLight 488 Microscale Antibody Labeling Kit References 1. Larsson, L. (1988). Immunocytochemistry: Theory and Practice. CRC. Boca Raton, 77-83, 224-225. 2. Goding, J. (1986). Monoclonal Antibodies: Principles and Practice, 2nd ed. Academic, London. General Reference 1. Horisberger, M. (1984). In Immunolabeling for Electron Microscopy. Polak, J., Varndel, I. Ed. Elsevier: Amsterdam Slide-A-Lyzer® Dialysis Cassette Technology is protected by U.S. Patent # 5,503,741 and 7,056,440. This product (“Product”) is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale, as set forth in the Product documentation, specifications and/or accompanying package inserts (“Documentation”) and to be free from defects in material and workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper and intended usage. 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