pHAb Reactive Dyes

pHAb Amine Reactive Dye

pHAb Reactive Dyes are available in two reactive forms suitable for antibody conjugation: pHAb Amine Reactive Dye and pHAb Thiol Reactive Dye.

pHAb Amine Reactive Dye has a succinimidyl ester group that reacts with primary amines available on the lysine amino acids on the antibodies.

pHAb Thiol Reactive Dye

pHAb Thiol Reactive Dye has a maleimide group that reacts with thiols. This maleimide group is conjugated to the antibody after the cysteine disulfide bonds in the hinge region of the antibody are reduced to thiols through the use of a reducing agent, such as DTT or TCEP. pHAb Reactive Dyes retain their fluorescent response to decreases in pH even after conjugation to antibody.

Monitor Antibody Internalization

A key feature of pHAb Dyes is that they have two sulfonate groups per dye, which increases solubility and reduces the aggregation often seen with other non-sulfonated dyes. pHAb Dyes maintain their fluorescence response to pH change even after antibody conjugation.

Even though antibody conjugation is the key application, any protein containing primary amines on lysine amino acids or thiols on cysteine amino acids can be conjugated with pHAb Dyes.

pHAb Reactive Dyes Retain Fluorescent Response to pH after Conjugation

pHAb Dye-conjugated antibodies can be used for monitoring receptor-mediated antibody internalization. When an antibody-pHAb Dye conjugate binds to its receptor on the cell membrane, it exhibits minimal fluorescence. However, upon receptor-mediated internalization, antibody-pHAb Dye conjugates traffic to the endosome and lysosomal vesicles where pH is acidic, causing the pHAb Dye to fluoresce. This fluorescence can be detected using various techniques, including cell imaging, flow cytometry and fluorescent plate-based readers with appropriate filters.