PAB-pHAb IgG Labeling Kit

PAB protein (Protein of Antibody Binding, MW 25 kDa) contains the IgG Fc-binding domain derived from Protein G and includes a cysteine residue at its C-terminus. Under reducing conditions, the C-terminal cysteine of PAB becomes a reduced thiol group. Through appropriate coupling reactions, toxins or dyes, including pH-sensitive dyes, can be specifically conjugated to this reduced thiol group. Such site-specific conjugation does not interfere with the specific binding between PAB and the IgG Fc region. PAB conjugated with toxins or dyes, including pH-sensitive dyes, can serve as a universal reagent for binding human or mouse IgG antibodies, enabling the study of antibody internalization or phagocytosis in relevant cells.

pHAb dyes are pH-sensitive fluorescent dyes that exhibit very low fluorescence at pH > 7, but their fluorescence significantly increases when the solution becomes acidic. The excitation wavelength (Ex) of pHAb dyes is 532 nm, and the emission wavelength (Em) is 560 nm; their fluorescence can be detected using TAMRA or Cy3 filters.

The PAB-pHAb IgG labeling kit includes the PAB-pHAb reagent, which is a complex in which pHAb is specifically conjugated to PAB via a reductive thiol group at the C-terminus of PAB. PAB-pHAb can serve as a universal binding reagent for human or mouse IgG antibodies, and it reflects the endocytic or phagocytic efficiency of antibodies by detecting the fluorescence emitted from the antibody-PAB-pHAb complex under the acidic conditions of endosomes or lysosomes. The PAB-pHAb IgG labeling reagent employs a unique method to remove free, unlabeled pHAb, thereby significantly reducing background caused by free pHAb. It is easy and quick to use, allowing antibody labeling to be completed within 30 minutes; the labeled antibodies do not require purification and can be directly used in subsequent intracellular endocytosis or phagocytosis experiments.

PAB-pHAb IgGThe specifications of the labeling kit are as follows. Among them, labeling…IgGQuantity according toPAB MW 25 kDa,IgGAntibodyMW 150 kDa, the molar ratio of the two markersFor5:1Calculation.

Specifications	Reagent volume	Reagent concentration (withPABCalculation)	Can be labeledIgG
250muL	250μL	250 μg/mL	seventy-fiveμg
1.2 ml	1.2 mL	250mugmL	350μg

According toPAB MW 25 kDaandIgGAntibodyMW 150 kDaCalculation,IgGandPAB-pHAbMolar ratio1:3To1:5Labeling can meet the needs of most experiments, and the molar ratio can also be optimized according to the specific experimental requirements, up to a maximum of10:1, which can enhance the detection signal strength. The following experiments useTrastuzumab andPAB-pHAbBy molar ratio1:5Detection after complex formationTrastuzumabIn expressionHER-2ofSK-BR-3The endocytic effect of cells.

1)At96For black, transparent cell culture plates.McCoy's 5A (10% FBS)Inoculate each well of the culture medium.100muL,20,000oneSK-BR-3cell,37°C 5% CO₂ Incubated overnight in an incubator.

2)Preparation10xTest concentrationTrastuzumab-PAB-pHAbComplex: As required by the experimentTake an appropriate amountOpti-MEM IDilute in culture mediumTrastuzumabto55μg/mL. To100μLof55μg/mL Trastuzumabadd18μLof250μg/mL PAB-pHAb, thusthe concentrations ofTrastuzumabandPAB-pHAb(10x)are approximately300 nMand1500 nM, mix well by pipetting, and incubate at room temperaturefor 30 minutes.It is recommended that after the first use ofPAB-pHAb, it be stored for a short term(2 weeks) at 4℃in the dark; for long-term storage, aliquot according to single-use amounts and store in the darkat -20℃or below

3)Take outSK-BR-3experimental cell plates, and add to each well10μLof the above-preparedTrastuzumab-PAB-pHAbComplex, mix by low-speed shaking. According toTrastuzumabCalculationTrastuzumab-PAB-pHAbThe final concentration of the complex is30 nM

4)37°C 5%CO2Incubation in a shaker incubator allows for the setting of multiple time points for sampling, for example:0 hours,2hr,4hr,6hr,12hr,24hretc.; the optimal detection time should be optimized based on the specific experiment. It is generally recommended to incubate for at least2hrThen begin to assess the endocytosis efficiency.

5)Using instruments that perform fluorescence detectionFluorescenceDetection of fluorescence in cells is performed using instruments such as fluorescence microscopes, flow cytometers, high-content imaging systems, and fluorescence plate readers.pHAbof the dyeExampleFor532 nm,EmFor560 nm,Can be usedTAMRAorCy3Filter detection, specificallyEx/EmRefer to the instrument's user manual for settings. To reduce background noise and obtain better experimental results, it is recommended to discard the culture medium and replace it with… before performing the assay.PBSUse a buffer to reduce background; note that this step does not terminate the endocytic reaction.

Do not mix different batches.
Do not alter the volume of the test reagent without rigorous validation.
For research use only.