In Situ Detection of Protein Complexes and Modifications by Chemical Ligation Proximity Assay. Supporting Information.

Transcription

1 In itu Detection of Protein Complexes and Modifications by Chemical Ligation Proximity Assay Rui ong*, Esteban Roberts and Christopher Bieniarz Technology and Applied Research, Ventana Medical ystems, Inc E. Innovation Park Drive, Tucson, AZ 8555, United tates upporting Information Table of Contents 1. tructures of reagents used for antibody modification 2. ynthetic scheme for Conjugate A (Figure 1) 3. upplemental Data 3.1. Typical EC chromatograph of purification of Conjugate A (Figure 2) 3.2. Detection of A-tag as a hapten (Figure 3) 3.3. Activity of Acetylene for Click reaction in Conjugate B (Figure 4) 3.4. Determine conditions for complete cleavage of A-tag from antibody (Figure 5) 3.5. Preservation of haptenated antibody under the reducing conditions (Figure 6) 3.6. Detection of E-cad/p120 in T4D and MDA-MB-453 cells by PLA and CLiPA (Figure ) 3.. Control experiment of CLiPA for pegfr detection (Figure 8) 1

2 1. tructures of reagents used for antibody modification PDP-dPEG8- ester was purchased from Quanta BioDesign, Ltd. (Plain City,, UA) Cys-A-3 was obtained from Bio-ynthesis, Inc (Lewisville, TX, UA) Alkyne-PEG4- ester from Click Chemistry Tools (cottsdale, AZ, UA) 2

3 2. ynthetic scheme for Conjugate A PDP-PEG 8 - ester GAR-PEG 8 -PDP 2 Cys 2-pyridinethione (EC 343 nm ~8000 cm -1 M -1 ) Cys-A-3 GAR-PEG 8 --A-3 2 Antibody-peptide conjugate with disulfide cleavable linker Figure 1. ynthetic scheme for GAR-P 8 --A- 3 (Conjugate A) 3. upplemental Data 3.1. Typical EC chromatograph of purification of Conjugate A Figure 2. A typical EC chromatogram for purification of the GAR-A-tag peptide conjugate (Conjugate A). 3

4 3.2. Detection of A-tag as a hapten Figure 3. Detection of Ki6 protein in FFPE tonsil tissue by A-tag using IC and DAB chromogenic detection system Activity of Acetylene for Click reaction in Conjugate B Figure 4. Detection of CD20 in tonsil sections using GAM-PEG4-CC conjugate: (a) Cu(I)/TPTA ratio of 1:5, (b) Cu(I)/TPTA ratio of 1:3, and (C) negative control with no Cu(I) added. 4

5 3.4. Determine conditions for complete cleavage of A-tag from antibody Figure 5. Determination of DTT concentrations to achieve complete cleavage of A-tag: (a) no DTT, (b) 50mM DTT, (c) 5 mm DTT, and (d) 100 mm DTT added Preservation of haptenated antibody under the reducing conditions We used VETAA ptiview DAB IC detection kit for the IC detection of protein bcl-6 in FFPE tonsil tissues to demonstrate that antibody is unaffected under the reducing conditions for cleaving the hapten in CLiPA. pecifically, mouse-anti-bcl6 (Ventana ) was use as the primary antibody followed by adding ptiview Q Universal Linker which contains hapten Q labeled secondary antibodies. The reducing reagent (100 ul per slide) was then added under the same conditions as in CLiPA (the effective concentration of TCEP is 1/3 to 1/4 of the indicated concentration since there is about ul liquid existing on the slide). After which, ptiview RP Multimer was added followed by DAB chromogenic detection. As show in Figure 6, there is no visible difference between PB and TCEP treated slides, indicating that the reducing conditions in CLiPA is not causing the dissociation of the bound hapten. In addition, it has been established that the IgG structure is maintained both by disulfide bonds and by noncovalent interactions between the heavy chains and light-heavy chains. (Wang, W., et al. (2006) Antibody structure, instability, and formulation J. Pharm. ci. 96, 1 26). 5

6 Figure 6. Detection of bcl-6 in FFPE tonsil with different concentrations of TCEP (a) PB control, (b) 50mM TCEP and (c) 100 mm TCEP Detection of E-cad/p120 in T4D in MDA-MB-453 cells by PLA and CLiPA a b Figure. Detection of E-cad/p120 in T4D in MDA-MB-453 cells by (a) PLA, and (b) CLiPA. 6

7 3.. Control experiment of CLiPA for pegfr detection a b Figure 8. CLiPA with omission of chelated Cu(I) catalyst for phosphor EGFR detection in KBr3 cells (a) +EFG, and (b) -EGF.