BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3381

Peptides:

Sequences	Affinity Values
NIFASAD(1) GLVDVLF(1) MNYREIY(1) GLVDALY(1) MNRHIPA(1) GLFALERSK(1)	[0.31] [0.52] [0.43] [n.a.] [n.a.] [n.a.]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (common panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 30394584

Target Name:

Anti N-terminal appetite-regulating hormone[24-51] polyclonal antibody

Template Name:

N-terminal appetite-regulating hormone[24-51]

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

Ph.D.-7 phage display library (X7)

Affinity Measurement Method:

ELISA

Affinity Measurement Description:

The fraction of N-terminal specific antibodies obtained after purification was evaluated usingenzyme linked immunosorbent assay (ELISA). Biotinylated human ghrelin (Bachem, Bubendorf, Switzerland) was immobilized to streptavidin coated microtiter plates (StreptaWell, Roche, Basel, Switzerland) and affinity purified anti N-terminal antibodiesobtained from 5 µg of purified antibodies or unpurified (5 µg) were added. Binding was detected by secondary goat anti rabbit horseradish peroxidase (HRP)-conjugated antibodies (Merck Millipore, Darmstadt, Germany) diluted 1:5000 in 0.1% bovine serum albumin (BSA) and reagent 3,3',5,5'-tetramethylbenzidine (TMB) (Thermo Fisher Scientific, Waltham, MA, USA). The absorbance at 450 nm (A (405 nm)) was read using the Tecan Safire (Tecan, Grödig, Austria) microtiter plate reader. A (405 nm) values were preproduced from Figure 1B and shown.

Brief Description:

Briefly, antibodies (3.4 to 1.7 μg; decreasing towards the third selection round) were incubated with phage libraries (2e11 pfu) in 100 μl of PBS solution with shaking at 400 rpm for 2 h in first selection round, 1 h in the second and for 0.5 h in the third. The resulting mixture was added to protein G or protein A coated magnetic beads (Dynabeads Protein G/A, Life Technologies, Invitrogen) in 1 mL of 0.1% BSA/0.1% PBST and incubated with shaking (400 rpm) to affinity capture the formed antibody-phage complexes. In each successive round of selection protein G and protein A beads were used alternatingly. Unbound phages were removed with repeated washings using 0.1 % PBST and bound phages were eluted with 400 μL of 200 mM glycine-HCl buffer (pH 2.2) containing 0.4 M NaCl and 0.1% BSA with 10 min of vigorous shaking (650 rpm). The beads were removed and the eluate was immediately neutralized by adding 1 M Tris-HCl (pH 9.1).

BiopanningDataSet Comments:

Curator:

Zhongjie Tang

Curation Date:

2019-01-29

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