Specification Sheet: Ferrocene-NHS Ester - Fivephoton NHS ester specification sheet...cene-NHS ester...
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FIVEphoton Biochemicals 4907 Morena Blvd, Ste 1403 San Diego, CA 92117 www.fivephoton.com Tel: (800) 462-4507 International Tel: (858) 395-4026 [email protected] Specification Sheet: Ferrocene-NHS Ester Part No. HPT1002 Product Name Ferrocene-NHS Ester (Ferrocene Carboxylic-NHS Ester (FerroEM-sensor TM )) Size 10 mg, 50 mg, 100 mg, 1 g. Bulk Quantities also available. Catalog #, Storage HPT1002 Storage: RT, desiccate Formula C 15 H 13 FeNO 4 Molecular Weight 327.11 Purity >97% Solvent System DMF or DMSO Absorbance Lamda max : 438 nm, Extinction Coefficient = 230 M -1 cm -1 Description Ferrocene-NHS Ester (FerroEM-sensor TM ) is an electrochemical active reporter molecule that reacts with primary amines in proteins and oligonucleotides, and is detected with voltammetry. The NHS reactive group allows for conjugation to both alpha and epsilon amino groups in peptide chains, as well as to free amino and thiol groups in nucleotides. Using standard reactive amine conjugation techniques, an amide linage is generated between the epsilon-amino group in lysine and the NHS moiety. A similar reaction scheme applies for oligonucleotides. Conjugation of ferro- cene-NHS ester to the epsilon-amino group in lysine also provides a multi-carbon linker between the polypeptide backbone and the ferrocene reporter, which attenuates steric hindrance on inter- molecular interactions. The structure of ferrocene-nhs ester is provided below. Fe O N O O O Peptide General Conjugation Protocol: Precipitate your protein (or peptide), and/or dilute into a non-amine buffer at pH 8.3 (for example 10 mM Hepes, 150 mM NaCl, pH 8.3) to the highest concentration that your protein (peptide) remains soluble. If you are using cell lysates, add non-amine containing protease inhibitors. Centrifuge and sediment insoluble debris using a microcentrifuge at full speed for 15 min. Collect the supernatant. Perform the Ferrocene Ester conjugation reaction with the supernatant. Dissolve Ferrocene NHS Ester in DMSO at 100mg/ml, then aliquot it immediately into the peptide-buffer solu- tion at a 100-1000X molar excess of Ferrocene Ester relative to protein. If the solution does not contain proteases (for ex- ample a synthesized peptide), let the reaction proceed at RT with gentle shaking for 4 hrs. If you are targeting a protein ex- tracted from cells, use protease inhibitors and perform the reaction overnight at 4 o C with gentle shaking. Subsequently, quench the reaction with a glycine solution at a molar ratio of glycine to Ferrocene Ester at 100:1 for about 15 min. You can purify the protein-ferrocene conjugate using gel filtration, or desalt in a spin column, collecting the ferrocene-peptide in the void volume. The Ferrocene-linked peptide should attain a yellowish coloration. The peptide-ferrocene conjugate can also be followed at 438 nm as noted above corresponding to the peptide bond absorbance peak. Store the ferrocene-peptide at –20 o C. ©FIVEphoton Biochemicals, 2015 Page 1
Transcript of Specification Sheet: Ferrocene-NHS Ester - Fivephoton NHS ester specification sheet...cene-NHS ester...
FIVEphoton Biochemicals4907 Morena Blvd, Ste 1403
San Diego, CA 92117www.fivephoton.comTel: (800) 462-4507
International Tel: (858) [email protected]
Specification Sheet: Ferrocene-NHS EsterPart No. HPT1002
Product Name Ferrocene-NHS Ester(Ferrocene Carboxylic-NHS Ester (FerroEM-sensorTM))
Size 10 mg, 50 mg, 100 mg, 1 g. Bulk Quantities also available.Catalog #, Storage HPT1002 Storage: RT, desiccateFormula C15H13FeNO4Molecular Weight 327.11Purity >97%Solvent System DMF or DMSOAbsorbance Lamdamax: 438 nm, Extinction Coefficient = 230 M-1cm-1
Description Ferrocene-NHS Ester (FerroEM-sensorTM) is an electrochemical active reporter molecule thatreacts with primary amines in proteins and oligonucleotides, and is detected with voltammetry.The NHS reactive group allows for conjugation to both alpha and epsilon amino groups in peptidechains, as well as to free amino and thiol groups in nucleotides. Using standard reactive amineconjugation techniques, an amide linage is generated between the epsilon-amino group in lysineand the NHS moiety. A similar reaction scheme applies for oligonucleotides. Conjugation of ferro-cene-NHS ester to the epsilon-amino group in lysine also provides a multi-carbon linker betweenthe polypeptide backbone and the ferrocene reporter, which attenuates steric hindrance on inter-molecular interactions. The structure of ferrocene-nhs ester is provided below.
Fe O N
O
O
O
Conjugation Summary Ferrocene-NHS Ester (9.85 mg, 30.11 -oligonucleotideis dissolved in 800 microliters of 0.2 M sodium carbonate buffer (pH 9.5). The ester solution (400amino-oligonucleotide solution. The mixture is left for 16 hour at 4oC, after which it is chromatographed on a Sephadex G-25 column using de-ionized water/carbonate buffer (50/50) as eluent. The fraction with yellow color is dialyzed againstwater to remove excess salts and unreacted reagents, and then freeze-dried. The final product is stored in the refrigeratoruntil use.
Peptide General Conjugation Protocol: Precipitate your protein (or peptide), and/or dilute into a non-amine buffer at pH 8.3(for example 10 mM Hepes, 150 mM NaCl, pH 8.3) to the highest concentration that your protein (peptide) remains soluble.If you are using cell lysates, add non-amine containing protease inhibitors. Centrifuge and sediment insoluble debris using amicrocentrifuge at full speed for 15 min. Collect the supernatant. Perform the Ferrocene Ester conjugation reaction with thesupernatant. Dissolve Ferrocene NHS Ester in DMSO at 100mg/ml, then aliquot it immediately into the peptide-buffer solu-tion at a 100-1000X molar excess of Ferrocene Ester relative to protein. If the solution does not contain proteases (for ex-ample a synthesized peptide), let the reaction proceed at RT with gentle shaking for 4 hrs. If you are targeting a protein ex-tracted from cells, use protease inhibitors and perform the reaction overnight at 4oC with gentle shaking. Subsequently,quench the reaction with a glycine solution at a molar ratio of glycine to Ferrocene Ester at 100:1 for about 15 min. You canpurify the protein-ferrocene conjugate using gel filtration, or desalt in a spin column, collecting the ferrocene-peptide in thevoid volume. The Ferrocene-linked peptide should attain a yellowish coloration. The peptide-ferrocene conjugate can alsobe followed at 438 nm as noted above corresponding to the peptide bond absorbance peak. Store the ferrocene-peptide at–20oC.
©FIVEphoton Biochemicals, 2015 Page 1
FIVEphoton Biochemicals4907 Morena Blvd, Ste 1403
San Diego, CA 92117www.fivephoton.comTel: (800) 462-4507
International Tel: (858) [email protected]
Specification Sheet: Ferrocene-NHS EsterPart No. HPT1002
Oligonucleotide Conjugation Protocol
Ferrocene-NHS Ester (9.85 mg, 30.11 micromol) is dissolved in 1.0 milliliter of methyl sul-foxide and 3 micromole amino-oligonucleotide is dissolved in 800 microliter of 0.2 M sodiumcarbonate buffer (pH 8.5). The ester solution (400 microliter) is added to the amino-oligonucleotide solution. The mixture is left for 4hrs at RT or 16 hour at 4oC, after which it ischromatographed on a Sephadex G-25 column using de-ionized water/carbonate buffer(50/50) as eluent. The fraction with yellow color is dialyzed against water to remove excesssalts and unreacted reagents, and then freeze-dried. The final product is stored in the re-frigerator until use.
Conjugation Summary Ferrocene-NHS Ester (9.85 mg, 30.11 -oligonucleotideis dissolved in 800 microliters of 0.2 M sodium carbonate buffer (pH 9.5). The ester solution (400amino-oligonucleotide solution. The mixture is left for 16 hour at 4oC, after which it is chromatographed on a Sephadex G-25 column using de-ionized water/carbonate buffer (50/50) as eluent. The fraction with yellow color is dialyzed againstwater to remove excess salts and unreacted reagents, and then freeze-dried. The final product is stored in the refrigeratoruntil use.
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©FIVEphoton Biochemicals, 2015 Page 2
