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Illustration for the expression and purification of S9.6 Fab-SoSi-His6 and <t>G3-SEAP</t> followed by SrtA-mediated conjugation. Two batches of ExpiCHO-S cells were used for the expression <t>of</t> <t>proteins.</t> (a) The first contained two cotransfected plasmids expressing S9.6-FabLC and S9.6-FabHC to produce S9.6 Fab, which was purified using a Ni-NTA column. (b) The second batch of cells contained the plasmid expressing G3-SEAP (SEAPd), which was purified through a hydroxyapatite column (Ceramic HTP, BioRad). The purified proteins were then conjugated using sortase A (SrtA) to generate Fab 1 –SEAPd and Fab 2 –SEAPd products. The conjugate products were then sequentially purified using Ni-NTA and hydroxyapatite column.

Seap Sequence, supplied by Addgene inc, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/pcdna3+3+ss+flag+ap+hndp/pcDNA3%2E3+ss-FLAG-AP-hNDP+(aa+25-133)+(Plasmid+%23115789)/pmc10197132-166-12-17
Average 91 stars, based on 1 article reviews

seap sequence - by Bioz Stars, 2026-10

91/100 stars

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1) Product Images from "S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids"

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

Journal: Bioconjugate Chemistry

doi: 10.1021/acs.bioconjchem.2c00609

Illustration for the expression and purification of S9.6 Fab-SoSi-His6 and G3-SEAP followed by SrtA-mediated conjugation. Two batches of ExpiCHO-S cells were used for the expression of proteins. (a) The first contained two cotransfected plasmids expressing S9.6-FabLC and S9.6-FabHC to produce S9.6 Fab, which was purified using a Ni-NTA column. (b) The second batch of cells contained the plasmid expressing G3-SEAP (SEAPd), which was purified through a hydroxyapatite column (Ceramic HTP, BioRad). The purified proteins were then conjugated using sortase A (SrtA) to generate Fab 1 –SEAPd and Fab 2 –SEAPd products. The conjugate products were then sequentially purified using Ni-NTA and hydroxyapatite column.

Figure Legend Snippet: Illustration for the expression and purification of S9.6 Fab-SoSi-His6 and G3-SEAP followed by SrtA-mediated conjugation. Two batches of ExpiCHO-S cells were used for the expression of proteins. (a) The first contained two cotransfected plasmids expressing S9.6-FabLC and S9.6-FabHC to produce S9.6 Fab, which was purified using a Ni-NTA column. (b) The second batch of cells contained the plasmid expressing G3-SEAP (SEAPd), which was purified through a hydroxyapatite column (Ceramic HTP, BioRad). The purified proteins were then conjugated using sortase A (SrtA) to generate Fab 1 –SEAPd and Fab 2 –SEAPd products. The conjugate products were then sequentially purified using Ni-NTA and hydroxyapatite column.

Techniques Used: Expressing, Purification, Conjugation Assay, Plasmid Preparation

$SrtA-mediated conjugation of purified S9.6 Fab-SoSi-His6 and G3-SEAP: (A) Elution profile of Ni-NTA-purified conjugation reaction run on a hydroxyapatite column. (B, C) Coomassie-stained SDS polyacrylamide gels with a reference ladder [lane L] and fractions from Figure A; (B) nonreduced and denatured proteins from fraction i contain SEAP (represented as denatured SEAPm); fraction ii includes a mixture of Fab 1 –SEAPm, SEAPm, and S9.6 Fab-SoSi-His6; and fraction iii includes purified Fab –SEAPd (shown as denatured Fab 1 –SEAPm). (C) Reduced and denatured proteins from fraction ii and fraction iii confirm the site-specific conjugation of G3-SEAP to S9.6 Fab HC (shown as Fab HC –SEAPm). Denatured SEAP (as SEAPm of 54 kDa) and S9.6 Fab LC (24 kDa) have resolved to their expected molecular sizes. (D, E) Confirmation of purified conjugates by SEC-MALS: elution profiles and molecular weights of purified (D) Fab 1 –SEAPd (145 kDa) and Fab –SEAPd (195 kDa) proteins and (E) SEAPd (110 kDa) protein.$
Figure Legend Snippet: SrtA-mediated conjugation of purified S9.6 Fab-SoSi-His6 and G3-SEAP: (A) Elution profile of Ni-NTA-purified conjugation reaction run on a hydroxyapatite column. (B, C) Coomassie-stained SDS polyacrylamide gels with a reference ladder [lane L] and fractions from Figure A; (B) nonreduced and denatured proteins from fraction i contain SEAP (represented as denatured SEAPm); fraction ii includes a mixture of Fab 1 –SEAPm, SEAPm, and S9.6 Fab-SoSi-His6; and fraction iii includes purified Fab –SEAPd (shown as denatured Fab 1 –SEAPm). (C) Reduced and denatured proteins from fraction ii and fraction iii confirm the site-specific conjugation of G3-SEAP to S9.6 Fab HC (shown as Fab HC –SEAPm). Denatured SEAP (as SEAPm of 54 kDa) and S9.6 Fab LC (24 kDa) have resolved to their expected molecular sizes. (D, E) Confirmation of purified conjugates by SEC-MALS: elution profiles and molecular weights of purified (D) Fab 1 –SEAPd (145 kDa) and Fab –SEAPd (195 kDa) proteins and (E) SEAPd (110 kDa) protein.

Techniques Used: Conjugation Assay, Purification, Staining

Coomassie-stained SDS polyacrylamide gel analysis of the purified S9.6 Fab–SEAP genetic fusion protein: the nonreduced lane shows the purified S9.6 Fab–SEAP genetic fusion protein as denatured Fab 1 –SEAPm (114 kDa). The reduced lane shows the Fab LC (25 kDa) and Fab HC –SEAPm (80 kDa) components of the S9.6 Fab–SEAP genetic fusion protein.

Figure Legend Snippet: Coomassie-stained SDS polyacrylamide gel analysis of the purified S9.6 Fab–SEAP genetic fusion protein: the nonreduced lane shows the purified S9.6 Fab–SEAP genetic fusion protein as denatured Fab 1 –SEAPm (114 kDa). The reduced lane shows the Fab LC (25 kDa) and Fab HC –SEAPm (80 kDa) components of the S9.6 Fab–SEAP genetic fusion protein.

Techniques Used: Staining, Purification

HC-S ELISA for the identification of DNA–RNA hybrid in solution. (A) Pictorial representation of the molecular interactions in the HC-S ELISA. The DNA–RNA hybrid is captured on S9.6 IgG-coated plates and subsequently detected using Fab 2 –SEAPd with chemiluminescence from the cleaved substrate as the readout. Image was created using Biorender.com. (B) The HC-S ELISA can specifically detect 100 pmole of DNA–RNA hybrid with as low as 5 pmole of Fab 2 –SEAPd (* indicates p > 0.05) in 100 μL of reaction. (C) Specific binding of 10 pmole of DNA–RNA hybrid, dsDNA, and dsRNA with 100 pmole of S9.6 Fab–LPET–SEAP genetic fusion and Fab 2 –SEAPd proteins in 100 μL of reaction measured in chemiluminescent units (A.U.). (D, E) HC-S ELISA is time- and temperature-dependent. The line graphs represent the signal output (as chemiluminescence in A.U. × 10 4 ) from HC-S ELISA tested at the mentioned assay times with different concentrations of DNA–RNA hybrids and 10 pmole of Fab 2 –SEAPd at (D) 4 °C and (E) 25 °C. * indicates p > 0.05 for statistical significance calculated using Student’s t -test.

Figure Legend Snippet: HC-S ELISA for the identification of DNA–RNA hybrid in solution. (A) Pictorial representation of the molecular interactions in the HC-S ELISA. The DNA–RNA hybrid is captured on S9.6 IgG-coated plates and subsequently detected using Fab 2 –SEAPd with chemiluminescence from the cleaved substrate as the readout. Image was created using Biorender.com. (B) The HC-S ELISA can specifically detect 100 pmole of DNA–RNA hybrid with as low as 5 pmole of Fab 2 –SEAPd (* indicates p > 0.05) in 100 μL of reaction. (C) Specific binding of 10 pmole of DNA–RNA hybrid, dsDNA, and dsRNA with 100 pmole of S9.6 Fab–LPET–SEAP genetic fusion and Fab 2 –SEAPd proteins in 100 μL of reaction measured in chemiluminescent units (A.U.). (D, E) HC-S ELISA is time- and temperature-dependent. The line graphs represent the signal output (as chemiluminescence in A.U. × 10 4 ) from HC-S ELISA tested at the mentioned assay times with different concentrations of DNA–RNA hybrids and 10 pmole of Fab 2 –SEAPd at (D) 4 °C and (E) 25 °C. * indicates p > 0.05 for statistical significance calculated using Student’s t -test.

Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

Affinity measurement using fluorescence polarization (FP) assay: 5′-FAM labeled DNA–RNA hybrid was used to measure FP-based affinity for S9.6 Fab (green), G3-SEAP (black), Fab 2 –SEAPd (red), and S9.6 Fab–SEAP genetic fusion protein (blue). N.D. indicates no interaction detected. Samples were run separately, and the resulting profiles are overlaid with their respective K d measurements included.

Figure Legend Snippet: Affinity measurement using fluorescence polarization (FP) assay: 5′-FAM labeled DNA–RNA hybrid was used to measure FP-based affinity for S9.6 Fab (green), G3-SEAP (black), Fab 2 –SEAPd (red), and S9.6 Fab–SEAP genetic fusion protein (blue). N.D. indicates no interaction detected. Samples were run separately, and the resulting profiles are overlaid with their respective K d measurements included.

Techniques Used: Fluorescence, FP Assay, Labeling

Figure Legend Snippet: List of Primers and Oligonucleotides

Techniques Used: Sequencing

Figure Legend Snippet: List of Clones and Expression Vectors Used in This Study

Techniques Used: Clone Assay, Expressing, Plasmid Preparation

other:

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids
Article Snippet: pcDNA3.3 ss–FLAG–AP–hNDP , expresses and secretes human-secreted alkaline phosphatase tags , Addgene #115789.

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Journal: Bioconjugate Chemistry

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

doi: 10.1021/acs.bioconjchem.2c00609

Figure Lengend Snippet: Illustration for the expression and purification of S9.6 Fab-SoSi-His6 and G3-SEAP followed by SrtA-mediated conjugation. Two batches of ExpiCHO-S cells were used for the expression of proteins. (a) The first contained two cotransfected plasmids expressing S9.6-FabLC and S9.6-FabHC to produce S9.6 Fab, which was purified using a Ni-NTA column. (b) The second batch of cells contained the plasmid expressing G3-SEAP (SEAPd), which was purified through a hydroxyapatite column (Ceramic HTP, BioRad). The purified proteins were then conjugated using sortase A (SrtA) to generate Fab 1 –SEAPd and Fab 2 –SEAPd products. The conjugate products were then sequentially purified using Ni-NTA and hydroxyapatite column.

Article Snippet: For the expression of fusion proteins of S9.6 Fab and SEAP, the SEAP sequence was excised from Addgene plasmid #115789 and was inserted into pcDNA3.4–S9.6 Fab HC using the NEBuilder HiFi DNA Assembly Cloning Kit (NEB) to create pcDNA3.4–S9.6 Fab HC –SEAP and pcDNA3.4–S9.6 Fab HC –LPET–SEAP.

Techniques: Expressing, Purification, Conjugation Assay, Plasmid Preparation

Journal: Bioconjugate Chemistry

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

doi: 10.1021/acs.bioconjchem.2c00609

Figure Lengend Snippet: SrtA-mediated conjugation of purified S9.6 Fab-SoSi-His6 and G3-SEAP: (A) Elution profile of Ni-NTA-purified conjugation reaction run on a hydroxyapatite column. (B, C) Coomassie-stained SDS polyacrylamide gels with a reference ladder [lane L] and fractions from Figure A; (B) nonreduced and denatured proteins from fraction i contain SEAP (represented as denatured SEAPm); fraction ii includes a mixture of Fab 1 –SEAPm, SEAPm, and S9.6 Fab-SoSi-His6; and fraction iii includes purified Fab –SEAPd (shown as denatured Fab 1 –SEAPm). (C) Reduced and denatured proteins from fraction ii and fraction iii confirm the site-specific conjugation of G3-SEAP to S9.6 Fab HC (shown as Fab HC –SEAPm). Denatured SEAP (as SEAPm of 54 kDa) and S9.6 Fab LC (24 kDa) have resolved to their expected molecular sizes. (D, E) Confirmation of purified conjugates by SEC-MALS: elution profiles and molecular weights of purified (D) Fab 1 –SEAPd (145 kDa) and Fab –SEAPd (195 kDa) proteins and (E) SEAPd (110 kDa) protein.

Techniques: Conjugation Assay, Purification, Staining

Journal: Bioconjugate Chemistry

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

doi: 10.1021/acs.bioconjchem.2c00609

Figure Lengend Snippet: Coomassie-stained SDS polyacrylamide gel analysis of the purified S9.6 Fab–SEAP genetic fusion protein: the nonreduced lane shows the purified S9.6 Fab–SEAP genetic fusion protein as denatured Fab 1 –SEAPm (114 kDa). The reduced lane shows the Fab LC (25 kDa) and Fab HC –SEAPm (80 kDa) components of the S9.6 Fab–SEAP genetic fusion protein.

Techniques: Staining, Purification

Journal: Bioconjugate Chemistry

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

doi: 10.1021/acs.bioconjchem.2c00609

Figure Lengend Snippet: HC-S ELISA for the identification of DNA–RNA hybrid in solution. (A) Pictorial representation of the molecular interactions in the HC-S ELISA. The DNA–RNA hybrid is captured on S9.6 IgG-coated plates and subsequently detected using Fab 2 –SEAPd with chemiluminescence from the cleaved substrate as the readout. Image was created using Biorender.com. (B) The HC-S ELISA can specifically detect 100 pmole of DNA–RNA hybrid with as low as 5 pmole of Fab 2 –SEAPd (* indicates p > 0.05) in 100 μL of reaction. (C) Specific binding of 10 pmole of DNA–RNA hybrid, dsDNA, and dsRNA with 100 pmole of S9.6 Fab–LPET–SEAP genetic fusion and Fab 2 –SEAPd proteins in 100 μL of reaction measured in chemiluminescent units (A.U.). (D, E) HC-S ELISA is time- and temperature-dependent. The line graphs represent the signal output (as chemiluminescence in A.U. × 10 4 ) from HC-S ELISA tested at the mentioned assay times with different concentrations of DNA–RNA hybrids and 10 pmole of Fab 2 –SEAPd at (D) 4 °C and (E) 25 °C. * indicates p > 0.05 for statistical significance calculated using Student’s t -test.

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Bioconjugate Chemistry

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

doi: 10.1021/acs.bioconjchem.2c00609

Figure Lengend Snippet: Affinity measurement using fluorescence polarization (FP) assay: 5′-FAM labeled DNA–RNA hybrid was used to measure FP-based affinity for S9.6 Fab (green), G3-SEAP (black), Fab 2 –SEAPd (red), and S9.6 Fab–SEAP genetic fusion protein (blue). N.D. indicates no interaction detected. Samples were run separately, and the resulting profiles are overlaid with their respective K d measurements included.

Techniques: Fluorescence, FP Assay, Labeling

Journal: Bioconjugate Chemistry

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

doi: 10.1021/acs.bioconjchem.2c00609

Figure Lengend Snippet: List of Primers and Oligonucleotides

Techniques: Sequencing

Journal: Bioconjugate Chemistry

Article Title: S9.6 Antibody–Enzyme Conjugates for the Detection of DNA–RNA Hybrids

doi: 10.1021/acs.bioconjchem.2c00609

Figure Lengend Snippet: List of Clones and Expression Vectors Used in This Study

Techniques: Clone Assay, Expressing, Plasmid Preparation

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seap sequence (Addgene inc)

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