Fab-pHast human is one of our fastest tools for quantitative testing of your primary antibody’s specificity, binding, and internalization, providing results in 1 day. Fab-pHast human binds to your primary human antibody via a secondary antibody cross-linked to a pH-dependent fluorescent reporter. This fluorescent reporter will increase intensity as the pH of its surroundings becomes more acidic, as evident when exposed to the environment inside a cell. A successful assay will provide an EC50 by way of a fluorescence detecting plate reader, illuminating your lead antibody candidates.
Fab-pHast human generates quantitative testing of the specific, binding, and internalization of your primary human antibody, with results in 1-day. This secondary conjugate is used to evaluate the potential of a primary antibody to internalize.
Fluorescent imagery of Fab-pHast Mouse conjugated to 192-IgG
HEK-293 cells, transfected with the p75 neurotrophin receptor, were plated at 20,000 cells/well in a 96-well plate and allowed to adhere overnight. 10 nM of the 192-IgG antibody (Cat. AB-N43) was incubated at room temperature with 30 nM of Fab-pHast Mouse (Cat. PH-02) for 20 minutes. The Fab-pHast conjugated antibody was then added to the cells and gently mixed for 2 min on a plate mixer. Cells were incubated overnight to allow internalization, although internalization could be detected within a few hours. Media was replaced with PBS to achieve a higher sensitivity and then the cells were analyzed on a fluorescent microscope under 20X magnification using a Y3 Leica filter cube.
*This data was acquired using Fab-pHast Mouse, Cat. #PH-02. We predict Fab-pHast Human, Cat. #PH-01, to behave similarly.
pHast Ab Internalization Assay
Parental HEK-293 cells, and HEK-293 cells transfected with the p75 receptor, were plated in a 96-well plate overnight. Titrated 192-IgG antibody (Cat. AB-N43) was incubated at RT with 50 nM of Fab-pHast Mouse (Cat. PH-02) for 20 minutes prior to addition to cells. Plates were incubated overnight to allow maximum internalization, but a few hours is sufficient for detection. Plates were read on a Spectra Max Gemini EM (Ex: 532nm/Em: 560nm). Data analysis was done by PRISM (GraphPad, San Diego).
To view protocol(s) for this and other products please visit: www.ATSbio.com/support/protocols