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In addition, the ribose of RNA is a reducing sugar. NHS ester-activated crosslinkers and labeling compounds react with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. Many purified enzymes and binding proteins of interest can be biotinylated and kept functional using this strategy if they are (1) glycosylated at sites away from functional domains and (2) not inactivated by treatment with periodate. Sodium cyanoborohydride (NaCNBH3) is a mild reducing agent that performs this function effectively without also reducing other chemical groups in biological samples. Lane 1: bacterial pellet, Lane 2: MW marker, Lane 3: culture supernatant, Lane 4: flow-through, Lanes 5-12: elutions, Lane 13: boiled resin following elutions. Comparison of several in vivo crosslinking methods. At concentrations of 6 to 10 mM periodate, other sugar groups in proteins will be affected. Glutaraldehyde (a bis-aldehyde), if added in large excess, can be used to aldehyde-activate the amines of one purified protein for conjugation by reductive amination to another protein.
A practical approach to crosslinking Mattson, G., et al.
Imidoester reaction scheme for chemical conjugation to a primary amine. There are at least two reasons for this popularity, availability and widespread use in labeling applications: To generate the he fluorescent western blot data that follows, a fluorophore conjugated secondary antibody was used to detect the target protein, ERK1 in cancer cell lysate.
Therefore, the more stable and efficient NHS-ester crosslinkers have steadily replaced them in most applications. AminoLink Resin and AminoLink Plus Resin are carefully prepared varieties of aldehyde-activated agarose resin. For example, Traut's Reagent (2-Iminothiolane, 2-IT) and SATA will add sulfhydryl groups onto primary amine sites.
Formaldehyde-treated and NHS-ester-treated cells were quenched with 100 mM glycine pH 3 and 500 mM Tris pH 8.0, respectively for an additional 15 mins.
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