Antibody internalization
Compare target-positive and target-negative samples over time. Assess antibody binding activity alongside fluorescence to evaluate receptor-mediated internalization.
pHrodo Labeling Series
For antibody internalization, cellular uptake and acidification studies.
pH560, pH630 and pHAb: 6 reactive dyes and 1 companion labeling reagent.
Fluorescence increases with acidificationMechanism schematic
- Binding
- Internalization
- Acidification
Product selection
Choose pHAb NHS ester or pH560/pH630 TFP ester for primary amines, and maleimide for free thiols. Signal increases with acidification, so consider sample pH as well as the band. Select the companion anti-human reagent separately for antibody compatibility.
6 products
| Product / Cat. No. | Product form | Excitation / nm | Emission / nm | Pack sizes | Product details |
|---|---|---|---|---|---|
| pH560 Red TFP Ester, Amine ReactiveQH-17-1005 | TFP Ester | 560 | 585 | 100 μg / 1 mg | |
| pH560 Red maleimide, Thiol ReactiveQH-17-1004 | Maleimide | 560 | 585 | 100 μg / 1 mg | |
| pH630 Deep Red TFP Ester, Amine ReactiveQH-17-1001 | TFP Ester | 640 | 655 | 100 μg / 1 mg | |
| pH630 Deep Red maleimide, Thiol ReactiveQH-17-1002 | Maleimide | 640 | 655 | 100 μg / 1 mg | |
| 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 |
Companion labeling reagent
A companion labeling option for human antibodies; see the product protocol for suitability.
| Product / Cat. No. | Excitation / nm | Emission / nm | Pack sizes | Product details |
|---|---|---|---|---|
| pH630 Deep Red anti-human labeling reagentQH-17-1003 | 640 | 655 | 200 μl |
pH response 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.
Assay design and interpretation
Uptake and acidification
Conjugate the probe to the molecule being tracked before detection. Use independent localization or acidification controls to distinguish uptake changes from changes in compartment acidity.
Multicolor detection
Select channels using actual excitation and emission peaks and filter bandwidths. Use single-color controls to assess spillover and keep gain settings consistent.
Labeling routes and applications
Complete handle-specific coupling and remove free dye, then compare the same conjugate under acidic and neutral reference conditions. Interpret cellular signal using target-negative samples, time courses, localization and acidification controls; fixation or permeabilization can alter compartment pH. Increased fluorescence alone neither quantifies absolute uptake nor demonstrates degradation.
Primary-amine labeling
TFP / NHS Ester · NH₂pH560 / pH630 TFP esters and pHAb NHS Ester. For antibodies or proteins with accessible primary amines; remove competing amines and carrier proteins before labeling.
Free-thiol labeling
Maleimide · SHpH560 / pH630 maleimides and pHAb thiol-reactive dye. Confirm free thiols, assess effects of reduction on protein structure and remove competing thiol reagents.
Antibody internalization
Compare target-positive and target-negative samples over time. Assess antibody binding activity alongside fluorescence to evaluate receptor-mediated internalization.
Uptake and acidification
Conjugate the probe to the molecule being tracked before detection. Use independent localization or acidification controls to distinguish uptake changes from changes in compartment acidity.
Multicolor detection
Select channels using actual excitation and emission peaks and filter bandwidths. Use single-color controls to assess spillover and keep gain settings consistent.
Technical 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.
Sample setup and controls for pH630 human-antibody labeling
Check the QH-17-1003 liquid formulation, antibody compatibility and background controls without applying a free-dye workflow.
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.
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.
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
Choose a document and enter your phone and email. Include the product lot number when requesting a CoA.
pH560 Red TFP Ester, Amine Reactive
QH-17-1005
pH560 Red maleimide, Thiol Reactive
QH-17-1004
pH630 Deep Red TFP Ester, Amine Reactive
QH-17-1001
pH630 Deep Red maleimide, Thiol Reactive
QH-17-1002
pHAb Amine Reactive Dye
QH-17-2001
pHAb Thiol Reactive Dye
QH-17-2002
pH630 Deep Red anti-human labeling reagent
QH-17-1003
Frequently asked questions
How do I choose between pH560, pH630 and pHAb?
Start with the instrument channels, then the labeling target. Excitation/emission wavelengths are 560/585 nm for pH560, 640/655 nm for pH630 and 532/560 nm for pHAb. Then choose chemistry for primary amines or free thiols.
Read the technical guide →Which samples suit TFP, NHS Ester and maleimide dyes?
TFP and NHS Esters label accessible primary amines on antibodies or proteins. Maleimides label accessible free thiols. Before choosing thiol labeling, check available thiols and whether reduction would affect structure or binding.
Read the technical guide →Can these dyes be added directly to cells?
The six reactive dyes first label the target molecule, followed by removal of free dye. The anti-human reagent is first mixed with a test human antibody using its dedicated workflow. These products are not general intracellular pH probes for direct addition to cells.
How does the anti-human reagent differ from a free pH630 dye?
The anti-human reagent uses a dedicated human-antibody suitability and mixing workflow; its name does not establish compatibility with every subclass or fragment. Free pH630 TFP/maleimide instead covalently labels amines or thiols and requires free-dye removal. Establish sample identity and route before comparing bands.
Read the technical guide →Can all products use the same preparation solvent?
No. Use anhydrous DMSO or DMF for pH560 and pH630 reactive dyes, and DMSO/water (1:1) for pHAb. The anti-human reagent is a liquid formulation and needs no reconstitution.
Read the technical guide →Which buffer additives should be checked for each labeling route?
For TFP/NHS, check reactive components such as Tris, glycine and BSA; ordinary buffer exchange does not reliably remove carrier proteins. For maleimide, confirm free thiols and address residual reductant after any reduction. Follow dedicated formulation requirements for anti-human rather than free-dye procedures.
Read the technical guide →Can all products be stored at −20°C?
Requirements differ. Store pH560 and pH630 reactive dyes dry and protected from light at −20°C. Store the anti-human liquid at −20°C, protected from light. Dry pHAb dye can be stored at −30°C to −10°C for 1 month; long-term storage requires below −65°C.
Does brighter fluorescence mean more internalization?
Local pH, labeling density and cell number all affect the signal. Record binding activity and labeling density, and use time courses, independent localization or uptake readouts and acidification controls. Fluorescence changes alone do not quantify absolute uptake or demonstrate degradation.
Read the technical guide →Can I fix or permeabilize the cells after staining?
Do not assume that processing preserves the original pH readout. Fixation, permeabilization and medium exchange can alter the probe environment. Measure live cells when studying live-cell internalization; if fixation is required, first compare response and localization before and after processing.
Read the technical guide →Can fluorescence intensity be converted directly to intracellular pH?
Not directly. Single-channel intensity depends on both probe amount and acidity. Absolute pH measurements require an appropriate reference readout and calibration in the actual assay; a product response curve does not replace cellular calibration.
Read the technical guide →What should I check first when signal is weak?
Check channels and acquisition settings, then the conjugate’s response under acidic conditions. If that response is normal, check post-labeling binding, target expression and cell health. This separates probe and instrument issues from cellular assay issues.
Read the technical guide →Need instructions for your product?
Request the manual with your antibody format and assay details.