Product manual
Product applications, preparation, use and storage requirements.
pH630 Deep Red overviewThiol-reactive dye
pH630 Deep Red maleimide, Thiol Reactive
Cat. No. QH-17-1002
Link proteins through free thiols and follow far-red fluorescence changes as labeled molecules reach acidic compartments.
Packs & quotepH630 Deep Red Maleimide
+Antibody / protein
→Labeled conjugate
Excitation640 nm
Emission655 nm
Labeling targetSH
Preparation and storage
- Preparation
- Anhydrous DMSO or DMF; prepare immediately before use
- Storage
- Store dry at −20°C, protected from light; avoid repeated freeze–thaw cycles
Applications 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.
- 1Cell-surface bindingCell membrane
- 2Internalization
- 3Acidic compartmentAcidic endosome / lysosome
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.
Specifications
Product information
- Product name
- pH630 Deep Red maleimide, Thiol Reactive
- Cat. No.
- QH-17-1002
- Product form
- Maleimide
- Labeling target
- Accessible free thiols
- Excitation / emission wavelengths
- 640 / 655 nm
- Pack sizes
- 100 μg / 1 mg
Preparation and properties
- Preparation solvent
- Anhydrous DMSO or DMF; prepare immediately before use
- Storage
- Store dry at −20°C, protected from light; avoid repeated freeze–thaw cycles
- Molar extinction coefficient
- 140,000 M⁻¹ cm⁻¹; in 4.3% (w/w) phosphoric acid
- Approximate pKa
- Approximately 5
- A280 / A260 correction factors
- 0.33 / 0.14; measured in 4.3% (w/w) phosphoric acid
- Storage stability
- At least 6 months at −20°C under the specified storage conditions
Fluorescence depends on the detection environment. Use product-specific parameters and matching measurement conditions for absorbance correction and labeling-density calculations.
Protocol
Use the product manual to set reagent amounts, reaction conditions and purification methods.
01Check free thiols and reduction conditions
Check whether accessible free thiols are already present. If reduction is needed, assess effects on protein structure and binding. Remove competing thiols such as DTT and check compatibility of other reducing agents.
02Prepare dye immediately before use
Let the sealed vial reach room temperature before opening. Prepare in anhydrous DMSO or DMF, protect from light and moisture, and use promptly.
03Control labeling density
Set buffer and reagent amounts for the selected chemistry. Compare labeling density, protein recovery and binding activity at small scale before scaling up; higher labeling density does not necessarily improve the assay.
04Remove free dye
Use a purification method appropriate for the target molecule’s size and confirm removal of unbound dye. Retain unlabeled protein as a binding and background control.
05Verify the conjugate’s pH response
Compare neutral and acidic conditions at matched concentration and acquisition settings before proceeding to cell assays. Record medium composition, sample state and labeling density.
Request product documentation
Choose a document and enter your phone and email. Include the product lot number when requesting a CoA.
pH630 Deep Red maleimide, Thiol ReactiveQH-17-1002
Certificate of analysis (CoA)
Test items and results for the corresponding lot.
Safety data sheet (SDS)
Safe handling, storage and emergency-response information.
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.
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.
Frequently asked questions
What can this dye label?
pH630 Deep Red maleimide, Thiol Reactive labels accessible free thiols. The presence of cysteine does not by itself establish that reactive free thiols are available.
Read the technical guide →Can the free dye measure intracellular pH directly?
This product is for preparing labeled conjugates, not a general intracellular pH probe for direct addition. Conjugate the target and remove free dye before following its acid-dependent signal.
Read the technical guide →Does pH630 require 630 nm excitation?
630 is the family name. The listed excitation/emission wavelengths are 640/655 nm. Check the actual laser line and far-red emission filter rather than choosing a channel from the name alone.
Read the technical guide →Which solvent should I use? Is water alone suitable?
Use anhydrous DMSO or DMF. Let the sealed vial reach room temperature before opening, then prepare and use promptly. Do not substitute water for the specified stock solvent.
Read the technical guide →Must I reduce the antibody, and can DTT remain in the reaction?
Do not automatically reduce a sample that already has accessible free thiols. If reduction is needed, assess its effect on structure and binding. Thiol-containing reducing agents such as DTT compete for maleimide and should be removed before conjugation; check compatibility of other reducing agents too.
Read the technical guide →How should the unreconstituted dye be stored?
Store dry at −20°C, protected from light; avoid repeated freeze–thaw cycles. Allow the sealed vial to reach room temperature before opening to limit moisture exposure. Follow the instructions for prepared solutions rather than applying the dry-dye shelf life.
Should I increase the amount when signal is weak?
First check the channel and acidic response of the purified conjugate. If the response is normal, assess post-labeling binding, target expression and cell health. Increasing the amount can also raise nonspecific background.
Read the technical guide →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 →Need instructions for your product?
Request the manual with your antibody format and assay details.