NHS Ester · Primary amines
A water-soluble NHS Ester for antibody primary-amine labeling and receptor-mediated antibody internalization studies.
ProtocolpHAb
Water-soluble NHS Ester and Maleimide dyes for antibody primary-amine or free-thiol labeling and internalization studies.
2 reactive dyes
Product selection
2 matching products
| Product / Cat. No. | Chemistry | Excitation / nm | Emission / nm | Pack sizes | Product details |
|---|---|---|---|---|---|
| pHAb Amine Reactive DyeQH-17-2001 | NHS Ester | 532 | 560 | 250 μg / 1 mg | |
| pHAb Thiol Reactive DyeQH-17-2002 | Maleimide | 532 | 560 | 250 μg / 1 mg |
Spectra and detection
Fluorescence increases as the environment becomes acidic. Signal depends on labeling density, uptake, local pH and acquisition settings. Keep labeling density and instrument settings consistent across comparisons.
- Reference product
- pHAb Amine Reactive Dye QH-17-2001
- Excitation / emission
- 532 / 560 nm
- Detection channel
- Select channels for the instrument’s filter bandwidths and assess spillover with single-color controls.
Applications & methods
pHAb NHS Ester
+Antibody / protein
→Labeled conjugate
Reference Cat. No. QH-17-2001
Maleimide · Free thiols
A water-soluble maleimide for labeling antibody free thiols and detecting acidic fluorescence after internalization.
ProtocolTechnical articles
pHrodo selection for antibody internalization
Compare detection channels and labeling routes for pHAb, pH560 Red, pH630 Deep Red and the human-antibody companion reagent, with channel and sample considerations for internalization assays.
NHS Ester and maleimide antibody labeling
NHS Esters label primary amines, while maleimides label free thiols. Choose a route based on available functional groups, formulation and reduction requirements, then verify the labeled antibody's function.
Antibody DOL calculation and evaluation
Learn how to calculate antibody degree of labeling from A280 and dye absorbance, apply the correct extinction coefficients, and troubleshoot free dye, incorrect corrections, and excessive labeling. Evaluate the result against performance in the intended assay.
Controls and readouts for pHrodo internalization assays
Separate target-mediated uptake, nonspecific uptake and acidification, and define what the fluorescence readout measures.
Troubleshooting pHrodo background and weak signal
Localize the problem through cell-free response, binding and cellular readouts before increasing dye or antibody amounts.
References
Methods and application studies using the reagents identified in each paper; these are not validation data for Qihe products.
Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye
Nath N, et al. · J Immunol Methods · 431:11–21 · 2016
Compares amine and thiol labeling and the effects of dye-to-antibody ratio on internalization assays.
DOI: 10.1016/j.jim.2016.02.001
Study details and application
- Study system
- Trastuzumab (HER2) and cetuximab (EGFR), labeled with pHAb and measured by plate fluorescence.
- Application to assay design
- Useful for comparing labeling routes alongside binding activity. Reported ratios belong to the studied antibodies and dyes, not to every product.
Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores
Liao-Chan S, et al. · PLoS One · 10(4):e0124708 · 2015
Tracks antibody internalization by separating surface and intracellular fluorescence, without relying on acid-dependent brightening.
DOI: 10.1371/journal.pone.0124708
Study details and application
- Study system
- Alexa Fluor-labeled anti-EphA2 antibodies with anti-fluorophore antibodies that quench surface fluorescence.
- Application to assay design
- Provides a complementary approach to separating uptake from acidification. Its quenching antibodies target Alexa fluorophores, not pHrodo dyes.
Avelumab internalization by human circulating immune cells is mediated by both Fc gamma receptor and PD-L1 binding
Jin H, et al. · Oncoimmunology · 10(1):1958590 · 2021
Separates contributions from Fcγ-receptor binding and PD-L1 binding to antibody internalization.
DOI: 10.1080/2162402X.2021.1958590
Study details and application
- Study system
- pHrodo-labeled avelumab and antibody variants, studied in human blood immune cells.
- Application to assay design
- In immune-cell assays, consider Fc-mediated uptake before assigning all signal to the target. Fc blocking depends on the biological question.
Request product documentation
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pHAb Amine Reactive Dye
QH-17-2001
pHAb Thiol Reactive Dye
QH-17-2002
FAQ
How do I choose a reactive form?
Choose a form for accessible primary amines or free thiols on the target. Check the buffer and additives before labeling and follow the manual for the exact catalog number.
Labeling routes and sample preparationHow do I request instructions and lot documentation?
Use the Documents tab to request the manual, SDS or CoA by catalog number. Include the vial lot number for a CoA.