PAB-MMAE IgG Labeling Kit

PAB protein(Antibody-binding protein, MW 25 kDa)Contains IgG derived from Protein G.FcIt contains a conjugation domain and a cysteine residue at the C-terminus; under reducing conditions, the C-terminal cysteine of PAB is converted into a reduced thiol group, allowing for the site-specific conjugation of toxins and dyes, including pH-sensitive dyes, via appropriate coupling reactions.ThisOn the reductive thiol group, such site-specific conjugation does not affect PAB.Specificityand IgGFcThe conjugation of PAB with toxins or dyes, including pH-sensitive fluorescent dyes, enables its use as a universal binding reagent for human or mouse IgG antibodies, facilitating the study of antibody internalization or phagocytosis in the corresponding cells.

Monomethyl auristatin E (MMAE) is a synthetic derivative of dolastatin 10 that exerts potent mitotic inhibition by inhibiting tubulin polymerization., exhibiting universal cell proliferation inhibition and/or cytotoxic effects.AtAntibody-Drug Conjugate (ADC)In the middle,MMAEis the mostWidespreadshoulduseofCytotoxicOne of the basics.

PAB-MMAE IgG Labeling KitIncluding the PAB-MMAE reagent, i.e., VcMMAE via PAB. Site-specific conjugation of a reducing thiol at the C-terminusThe complex on PAB. The conjugation between PAB and MMAE is achieved via the currently most widely used cleavableLinker mc-VC-PABCachieveThe linker comprises a maleimide-based spacer, the Val–Cit dipeptide sequence serving as a substrate for cathepsin, and a PABC self‑cleaving spacer..PAB-MMAEIt can serve as a universal binding reagent for human or mouse IgG antibodies., by detectionAntibody-PAB-MMAE conjugateIn the corresponding cellThe cytotoxic effect is used to reflect the antibody.Endocytic or phagocytic effects. PAB-MMAE IgG labeling reagent.It is simple and fast to use, allowing antibody labeling to be completed within 30 minutes. The labeled antibody does not require purification and can be directly used in subsequent cellendocytosis or phagocytosisexperiments. Moreover, compared with commonly used conjugated toxins such as DT3C, the PAB-MMAE reagent has a smaller molecular weight, which reducesthe binding of PAB to IgG antibodiesand thus minimizes the potential impact on antibody function,while the VC-PABC linkerbetter simulates the actual application of ADCs.

PAB-MMAE IgGThe specifications of the labeling kit are as follows. Among them, the labelingIgGquantity is based onPAB MW 25KDa,IgGantibodyMW 150KDa, and the molar ratio for their labeling isis3:1calculated.

Specifications	Reagent volume	Reagent concentration (PAB)	Can labelIgG
50μg	2x50μL	250μg/mL	50μg

According toPAB MW 25 kDaandIgGAntibodyMW 150 kDaCalculation,IgGandPAB-MMAEBy molar ratio3:1Labeling 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 of6:1, which can enhance the detection signal strength. The following experiments useTrastuzumab andPAB-MMAEExpression of the complex formed by combinationHER-2ofSK-BR-3The cytotoxic effect of cells is used to reflect the endocytic efficacy of antibodies.

1)At96For white transparent cell culture plates.McCoy's 5A (10% FBS)Inoculate each well of the culture medium.100mul,4,000oneSK-BR-3cell,37°C 5% CO₂ Incubated overnight in an incubator.

2)Preparation10xTest concentrationTrastuzumab-PAB-MMAEComplex: As required by the experimentTake an appropriate amountOpti-MEM Idilute the mediumTrastuzumabto50μg/mL, then according to10:1addPAB-MMACuntil the concentration reaches22.7μg/mL, for example,30μLof50μg/mL Trastuzumabadd3μL PAB-MMAC, so thatthe concentrations ofTrastuzumabandPAB-MMAC(10x) are approximately300 nMand900 nM, mix by pipetting, and incubate at room temperaturefor 30 minutes.It is recommended that after the first use ofPAB-MMAE, it be stored short-term(2 weeks) at4℃, and for long-term storage, aliquot according to single-use amounts and store protected from lightat-20℃or below

3)UseOpti-MEM Imedium to perform3X serial dilutions on the above-preparedTrastuzumabandPAB-MMAEcomplex, resulting in a total of8concentrations

4)take outSK-BR-3Experimental cell plates, add to each well:10Gradient concentrations prepared in volumes of μL or greaterTrastuzumab -PAB-MMAEComplex, according toTrastuzumabThe calculated maximum final concentration is30 nM,3xDilute, total8a concentration

5)37°C 5%CO2Incubator incubation5Heaven, suggestion3Added due to favorable timing.20muL McCoy's 5A(10% FBS) Culture medium

6)to carry outCell viabilityDetection, For example, you can useUA-Glo® China Telecom GroupCell viability assay kit ( UA070103) Detect cell viability

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