pHAb Dyes are pH sensor dyes that have very low fluorescence at pH more than 7 and a dramatic increase in fluorescence as the pH of the solution becomes acidic. pHAb Dyes have excitation maxima (Ex) at 532nm and emission maxima (Em) at 560nm. pHAb Dyes are designed specifically for antibody labeling and are available in two reactive forms suitable for antibody conjugations. 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 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 using a reducing agent, such as DTT or TCEP. 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.|
Accurately Determine Antibody Internalization: Increase in fluorescence as the pH of the solution becomes acidic.
Conjugate Directly from Biological Samples on Expressing Antibodies (i.e., Cell Media): On-bead conjugation.
Measure Internalization in Real Time: Compatible with 96-well, plate-based assay.
Know that Antibody Conjugated with pH-Sensitive Dye is Fluorescent Only when Internalized: pH profile of free and antibody conjugated dye is similar.
Get High Signal-to-Background Ratios: Individual dyes are cell-impermeable when unconjugated.