TFP Ester · Primary amines
Label antibody or protein primary amines and follow uptake and acidification through the conjugate’s increased fluorescence at acidic pH.
ProtocolpH560 Red
TFP Ester and Maleimide forms for antibody and protein labeling and acidification-associated internalization assays.
2 reactive dyes
Product selection
2 matching products
| Product / Cat. No. | Chemistry | Excitation / nm | Emission / nm | Pack sizes | Product details |
|---|---|---|---|---|---|
| pH560 Red maleimide, Thiol ReactiveQH-17-1004 | Maleimide | 560 | 585 | 100 μg / 1 mg | |
| pH560 Red TFP Ester, Amine ReactiveQH-17-1005 | TFP Ester | 560 | 585 | 100 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
- pH560 Red TFP Ester, Amine Reactive QH-17-1005
- Excitation / emission
- 560 / 585 nm
- Detection channel
- Select channels for the instrument’s filter bandwidths and assess spillover with single-color controls.
Applications & methods
pH560 Red TFP
+Antibody / protein
→Labeled conjugate
Reference Cat. No. QH-17-1005
Maleimide · Free thiols
Conjugate accessible free thiols to prepare pH-responsive probes through protein cysteine labeling.
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.
Intracellular Delivery and Trafficking Dynamics of a Lymphoma-Targeting Antibody–Polymer Conjugate
Berguig GY, et al. · Mol Pharm · 9(12):3506–3514 · 2012
Uses ratiometric fluorescence, imaging and flow cytometry to assess the acidity experienced by labeled molecules.
DOI: 10.1021/mp300338s
Study details and application
- Study system
- An HD39 antibody system in CD22-positive cells, dual-labeled with Alexa Fluor 488 and pHrodo.
- Application to assay design
- Useful for understanding reference channels and calibration. Neither single-channel intensity nor an uncalibrated ratio directly gives absolute pH.
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.
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.
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.
Request product documentation
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pH560 Red TFP Ester, Amine Reactive
QH-17-1005
pH560 Red maleimide, Thiol Reactive
QH-17-1004
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.