Product manual
Product applications, preparation, use and storage requirements.
pH630 Deep Red overviewHuman-antibody labeling reagent
pH630 Deep Red anti-human labeling reagent
Cat. No. QH-17-1003
A pH-responsive labeling reagent for human-antibody internalization studies, detecting fluorescence changes after entry into acidic compartments.
Packs & quoteLabeling reagent
+Human antibody
→Labeled complex
Excitation640 nm
Emission655 nm
Labeling targetHuman antibody
Preparation and storage
- Preparation
- Liquid formulation, 0.5 mg/mL
- Storage
- Store at −20°C, protected from light
Applications and detection
Fluorescence increases as the environment becomes acidic. Signal depends on antibody and reagent amounts, uptake, local pH and acquisition settings. Keep reagent ratios, sample amounts 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.
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 anti-human labeling reagent
- Cat. No.
- QH-17-1003
- Product form
- Human-antibody labeling reagent
- Labeling target
- Compatible human antibodies
- Excitation / emission wavelengths
- 640 / 655 nm
- Pack sizes
- 200 μl
Preparation and properties
- Formulation
- Liquid formulation, 0.5 mg/mL
- Storage
- Store at −20°C, protected from light
- Reagent concentration
- 0.5 mg/mL
- Buffer
- 10 mM PBS, pH 7.4, with 50% glycerol
Protocol
Use the dedicated manual to confirm antibody compatibility and labeling conditions.
01Check antibody compatibility
Confirm the human-antibody type, formulation components and assay compatibility, and follow this reagent’s specific labeling instructions.
02Establish binding and uptake controls
Include unlabeled antibody, target-negative samples and reagent blanks. Confirm that labeling preserves antibody binding before comparing internalization.
03Record a time course
Keep sample handling and acquisition settings consistent. Record fluorescence and intracellular localization over time, with acidification controls when needed.
Request product documentation
Choose a document and enter your phone and email. Include the product lot number when requesting a CoA.
pH630 Deep Red anti-human labeling reagentQH-17-1003
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.
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.
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.
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
Which antibodies can use this anti-human reagent?
This reagent is intended for human-antibody labeling. The name “anti-human” does not establish compatibility with every IgG subclass or fragment. Include species, subclass, antibody format and formulation when requesting information for a specific sample.
Read the technical guide →Can I add the labeling reagent directly to cells?
First mix it with the test antibody using the dedicated workflow, then prepare the cell-assay working solution. Include a reagent-only control without test antibody to assess reagent-associated background.
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 →Does the reagent need reconstitution?
No. It is supplied as 200 μL of liquid at 0.5 mg/mL. Mix gently without sonication and prepare the labeling mixture using its dedicated instructions. Do not use a preparation procedure for powdered TFP, NHS Ester or maleimide dyes.
Read the technical guide →Will the reagent introduce glycerol into the cell assay?
Yes. The stock contains 50% glycerol, so account for its final concentration after dilution. Keep reagent ratios and total volumes consistent when comparing antibodies, and use a matched reagent control to assess background and cell health.
Read the technical guide →How should the liquid reagent be stored?
Store at −20°C, protected from light, and avoid repeated freeze–thaw cycles. Keep opened reagent sealed as instructed. Stock storage conditions do not establish the shelf life of diluted working solutions.
Should I increase the amount when signal is weak?
First check the channel, antibody binding and cell health, and compare the complete mixture with a reagent-only control. More reagent may also increase background. Establish whether the issue is the antibody, labeling system or detection settings first.
Read the technical guide →Does brighter fluorescence mean more internalization?
Not necessarily. Local pH, antibody and reagent amounts, and cell number also affect the signal. Keep reagent ratios consistent and check binding activity when comparing antibodies. Add an independent localization or uptake readout to distinguish uptake from acidification.
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.