BDB -BiopanningDataSet Details

Biopanning Data

BiopanningDataSet ID: 3562

Peptides:

Sequences	Affinity Values
WHYNWQDVSDRQ HQNHHSTFWEIY	[4.1] [6.5]

Sequences Features:

Motif:

Unique Sequence Number:

Experimental Method:

Phage display (subtractive panning)

Sequencing Platform:

Sequencing Method:

Sanger sequencing

Rounds of Panning:

Reference:

PMID: 31301216

Target Name:

Fab A,Fab Z and Fab D

Template Name:

Not determined.

Structure of Target-Template Complex:

Not determined.

Structure of Target-Peptide Complex:

Not determined.

Library Name:

Ph.D.-12 phage display library (X12)

Affinity Measurement Method:

Fluorescence polarization

Affinity Measurement Description:

The labeled peptide was diluted in PBS to prepare a stock solution of 500 nM. Fab A was diluted in PBS to two different concentrations (2 and 20 μM). Concentrations of peptide and Fab A were measured using a NanoDrop UV Spectrophotometer (Harlow Scientific). The level of binding was evaluated using fluorescence polarization (FP) by mixing equal volumes (20 μL) of the peptide and each Fab concentration in a 384 well black plate, resulting in a final peptide concentration of 250 nM. The dissociation constants (KD, μM) were determined.

Brief Description:

The first round of screen was performed with Fab A as target. Amplified phage from the first positive was diluted to a concentration of e11 pfu/200 μL and incubated thrice (first negative) in: (a) BSA blocked tube, for 1 hr at RT; (b, c) protein L magnetic beads, without Fab, for 50 and 40 min, respectively, at RT. In each incubation, the supernatant of the previous step was used. The supernatant after the first set of negative rounds was collected and incubated with Fab Z immobilized on Protein L magnetic beads, for 1 hr at RT (second positive). All the amplified phage from the second positive (concentration of e11 pfu/200 μL) was used in a second negative selection round, similar to the first negative (second negative). The supernatant of the second negative was collected and incubated with Fab D immobilized in protein L magnetic beads, for 45 min at RT. The amplified phage from the third positive was diluted to a concentration of e11 pfu/200 μL and added to four different tubes containing NHS beads (fourth positive) with: (a) a mixture of the three used Fabs, (b) Fab A, (c) Fab Z, and (d) Fab D.

BiopanningDataSet Comments:

The top two peptide binders, A5 (WHYNWQDVSDRQ) and C7 (HQNHHSTFWEIY), were determined to have KD values of 4.1 μM and 6.5 μM, respectively.

Curator:

Chunchao Pu

Curation Date:

2020-12-29

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