DiTagTM

pH sensitive IgG labeling reagents

DiTag

pH sensitive IgG labeling reagents provide an easy solution to measure internalization activities

Catalog No:TMAME100001of antibodies. This reagent utilizes a pH-sensitive fluorescently labeled Fc binding protein that binds toCH

IgG antibodies from various species, resulting in the formation of a fluorescently labeled antibody-reagent
complex. After antibody internalization, the surrounding pH becomes acidic and significantly enhances

fluorescence signal of antibody-reagent complex. The fluorescence intensity can be used as an indicator
to determine the internalization activity of antibodies. By measuring the strength of the fluorescenceTEsignal, researchers can assess the efficiency of antibody internalization into cells. This information is

crucial in understanding the cellular uptake mechanism of antibodies and assessing their efficacy intargeted therapies or diagnostic applications. Additionally, monitoring the fluorescence intensity can also
provide insights into the kinetics of antibody internalization, helping researchers optimize experimental

DIMABIO

condition and improve the design of antibody-based drug delivery systems.

Table 1 Contents and storage
Description

Applications
Formulation
Storage

Excitation/emission maxima
Detection method
MWDiTagTM

pH sensitive IgG labeling reagent

Antibody internalization labeling

Lyophilized from 1xPBS (pH 7.4). Normally 5% - 8% trehalose is added as protectants

before lyophilization. Please see Certificate of Analysis for specific instructions.

The reagents are supplied in lyophilized form. We recommend storing the vial(s) at -20℃, desiccated and protected from light. Once reconstituted, the reagents can be stored

at 2-8℃ for 1~2 weeks, or with 50% glycerol at -20℃.
505/525 nm

Flow cytometry, detected with FITC or AF488 filter
The product has a MW of 33.4 kDa

For Research Use Only. Not for use in diagnostic procedures.
DIMA Biotechnology LTD

Telephone: 400-006-0995

Email: info@dimabio.com

Website: www.dimabio.com
1

Protocol : DiTagTM

pH sensitive IgG labeling reagents

1.Reconstitution of AME100001

Briefly centrifuge or tap down the vial of AME100001 to ensure that all product is collected at the bottomof the vial. Add the correct amount of sterile ddH2O to the vial to make the final concentration same asCH

what indicated on COA. Reconstitute AME100001 with gentle agitation.
2.Prepare antibody-AME100001 complex in 2× working solution

Before actual internalization cell assay, a 2× working solution for Antibody -AME100001 complex needs toratio (9:1 by mass calculation).

TEbe prepared first. For the best results, we recommend test antibody to mix with AME100001 at 2:1 in molar

Based on the final experimental setting, calculate the desired amount of test antibody and AME100001
stock solution (prepared in step1), mix thoroughly and add enough complete culture medium to makeO

100ul in final volume. Leave the 2× antibody-AME100001 complex solution at room temperature for 1hrand protected from light exposure by covering with aluminum foil.

BI

3.Internalization Assay

Prepare the cells at 1~ 2 × 10 cells/mL in cell culture medium.
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Add 100ul of cells to each well of a 96-well plate containing 100ul of 2X antibody-AME100001 working

solution from step 2.

DIMA

Incubate the cells with antibody-AME100001 complex at 37℃ for 18-24hours under standard cell culture

conditions.

The internalization efficiency of antibodies was measured using flow cytometry.

For Research Use Only. Not for use in diagnostic procedures.
DIMA Biotechnology LTD

Telephone: 400-006-0995

Email: info@dimabio.com

Website: www.dimabio.com
2

TM

pH sensitive IgG labeling reagentsTE

CH

Assay Data : DiTag

Figure1. The fluorescent signal from GPRC5D ADC BMK-AME100001 conjugate is only detected in GPRC5D

DIMA

BIO

positive cells (K562-GPRC5D stable expression cell line), indicating specific internalization.

Figure2. Stability test of AME100001. Three storage methods are tested: lyophilization and reconstitution,liquid with 50% glycerol at -20℃, liquid at 4℃. All three methods exhibit excellent stability.

Ordering information
Cat. No.

Product name

AME100001-50ug

DiTag

AME100001-10ug

AME100001-100ug

DiTag

TM

DiTag

TMTM

pH sensitive IgG labeling reagent

10ug

pH sensitive IgG labeling reagent

100ug

pH sensitive IgG labeling reagent

For Research Use Only. Not for use in diagnostic procedures.
DIMA Biotechnology LTD

Telephone: 400-006-0995

Unit size

Email: info@dimabio.com

Website: www.dimabio.com
350ug

FAQ

1. What is the MW of Fc binding protein in DiTag
The MW of Fc binding protein in DiTag

TMTM

pH sensitive IgG labeling reagent?

pH sensitive IgG labeling reagents is 33.4kDa.

2. How much antibody and cells should I use with this reagent?

When labeling an antibody with DiTagTM pH sensitive IgG labeling reagent, we suggest a molarCHratio of 1:2 reagent: antibody in the reaction. For example, if you are labeling a 150kDa candidate

antibody with DiTagTM reagent, you can use 1ug of reagent for 9ug of antibody and test antibody

internalization using 5000-10000 cells. To determine the optimal conditions for your antibody
candidates and cells, you should try different ratios and number of cells.

3. Why is fixation not recommended for cells after incubation of antibody-reagent complex?

TEThe pH-sensitive dye used in reagent in relatively non-fluorescent until it enters the acidic

endosome, at which point its fluorescence increases. They should only be used on live cells todetect pH of cellular compartments. Upon fixation of the cells, the cell membrane is compromised
and you are only measuring the pH of the buffer the samples are held in. For this reason, we do notrecommend fixing samples.
the reagent?

O

4. What is the fluorescence excitation/emission maxima for the pH-sensitive dye, provided inBI

The fluorescence excitation/emission maxima for the pH-sensitive dye is 505/525 nm.

5. Which IgG subtypes can we use with this reagent?

The DiTagTM pH sensitive IgG labeling reagents can be used for human IgG1 、 IgG2 and IgG4,rabbit IgG, mouse IgG2a and IgG2b.

6. Are there any specific requirements for the downstream applications? Can we use flowDIMAcytometry and/or Incucyte after labeling and antibody internalization?

We test antibody internalization using flow cytometry. Live cell dynamic imaging and analysis
system such as Incucyte has not been tested.

7. How to reconstitute the reagent? Can we apply the antibody-reagent complex directly to thecells? Is it sterile?

Deionized water is recommend for the reconstitution according to the COA. This reagent is notsterile, but wo have not observed cell contamination within 1-24 hours yet.

8. Is this reagent stable? Does it need to be stored in aliquot? How long can we store thisreagent?

The reagents are supplied in lyophilized form. We recommend storing the vial(s) at -20 ℃ ,desiccated and protected from light. Once reconstituted, the reagents can be stored at 2-8℃ for

1~2 weeks, or with 50% glycerol at -20℃.

For Research Use Only. Not for use in diagnostic procedures.
DIMA Biotechnology LTD

Telephone: 400-006-0995

Email: info@dimabio.com

Website: www.dimabio.com
4