Issue 9June, 2016

Isolation of drug target protein (4)Biotin-Streptavidin Binding assay 【Biotinylated MTX】
Introduction
Streptavidin is a protein that binds to biotin with a very strong affinity, and biotin is used as a low-molecular-weight compound that can beeasily and effectively introduced into compounds, proteins, nucleic acids, etc. as a label. For this reason, immobilization of biotinylated
ligands on streptavidin beads and purification experiments of target substances using them are being conducted in a wide range of research
fields.
There are two approaches to experiments with streptavidin beads, “The direct method” and “The indirect method”. The method of reacting theligand previously bound to the beads with the target is called the direct method, and the method of reacting the ligand with the target andthen binding the ligand-target complex to the beads is called the indirect method. Use these methods properly according to the purpose ofthe experiment.
In this study, chemical pull-down of DHFR by MTX (methotrexate) was performed using biotinylated MTX and streptavidin beads. In addition
to comparing performance with other companies' beads, it also shows the importance of the length of the spacer arm that connects MTX andbiotin. This is because streptavidin can cause steric hindrance, which can interfere with the binding of ligands to target proteins.

ResultBiotinylated MTX with different spacer arm lengths were immobilized on streptavidin beads, and DHFR was purified from HeLa cell lysate. As
a control, the same operation is performed for the MTX amino group derivative (without biotin) immobilized on NHS beads.
The following points can be confirmed from the two in the figure below.
・ FG beads have less non-specific adsorption than other companies' beads.
・ DHFR could not be purified with a spacer arm of about 20 Å, but could be purified with a spacer arm of about 30 Å. From this, it wasconfirmed that the length of the spacer arm should be set to about 30 Å or more when biotinlating the low molecular weight compound.

Biotinylated MTX (Spacer arm Length : about 20Å)

Biotinylated MTX (Spacer arm Length : about 30Å)

BiotinMTXMTXStreptavdin beads

NHS beads
FG beads(kDa)
200MIn-+FG beads

Competitor--+Biotin

Spacer arm

Spacer arm+Beads
Production
Immobilize MTX

Streptavdin beads

NHS beads(kDa)
200116
97.4

116
97.4

656545453131

M

InFG beads

FG beads

Competitor---+++Beads
Production
Immobilize MTX

DHFR

DHFR

21.5

21.5
14.4

Streptavidin

6.5

14.4

Streptavidin

6.5
Silver staining

TAMAGAWA SEIKI CO., LTD.

Silver staining

http://www.FGbeads.com

Materials
aterials and method
Mand method■Materials■FG beads® information

1. Streptavidine beads
2. Biotinylated MTX
3. PBS(-)(137mM NaCl, 2.7mM KCl, 8.1mM Na2HPO4・12H2O,1.5mM KH2PO4)
4. Wash buffer (10mM HEPES-NaOH(pH7.9), 50mM KCl,0.2mM EDTA, 10% glycerol)
5. HeLa cell extracts (cytosolic fraction) ― 3mg/ml
6. 100mM KCl buffer (20mM HEPES-NaOH(pH7.9), 100mM KCl,
1mM MgCl2, 0.2mM CaCl2, 0.2mM EDTA, 10% glycerol,
0.1% NP-40, 1mM DTT, 0.2mM PMSF)
7.SDS sample buffer (62.5mM Tris-HCl(pH6.8), 0.005% BPB,2% SDS, 10% glycerol, 5% 2-mercapto ethanol)

Product nameStreptavidin beads

Product number

TAS8848N1170

Storage temperature

2-8oC

Storage buffer

10mM HEPES(pH7.9), 50mM KCl,
1mM EDTA, 10%glycerol

Size of beads

190nm ± 20 nm
Free biotion >1200pmol

Binding capacity(for 1mg of beads)

Biotinylated Antibody >13ug
Biotinilated ssDNA (50mer) >13ug
Biotinylated BSA >3ug

■ Method 2 (Affinity Purification)■ Method 1 (Immobilize biotinylated MTX)
1. Immobilize biotinylated MTX on beads
Transfer 0.5mg beads to a tube, and wash beads with PBS(-)
3times at 4oC.
Add 0.1mM biotinylated MTX diluted in 200ul PBS(-) to beads,
and 200ul PBS(-) to another beads (Control beads).
Mix for 2hour at 4oC.
2. Wash
Wash biotinylated MTX immobilized beads and Control beads
with Wash buffer 3 times at 4oC, and storage with 200ul Wash
buffer.

1. Beads equilibration
Wash 0.5mg of each MTX immobilized beads with100mM KCl buffer 3 times at 4oC.
2. Reaction
Add 200ul HeLa cell extract and resuspend beads.
Incubate with rotation for 2hour at 4oC.
3. Wash
Separate magnetically and remove supernatant .
Wash beads with 100mM KCl buffer 3 times at 4oC.
4. Elution
Add 40ul of SDS sample buffer and resusupend beads.
Boil for 5min at 98oC, and remove beads.
5. Analyze the samples by SDS-PAGE and silver stainning.

■ Influence of Spacer Lengh on Steric hindrance
Short Spacer

BiotinMTXLong Spacer

Biotin×Streptavidin

DHFR

MTX has no accessibility for DHFRTAMAGAWA
SEIKI
CO.,
LTD.
TAMAGAWA
SEIKI
CO.,
LTD.

MTX ○
Streptavidin

DHFR

MTX has accessibility for DHFR

http://www.FGbeads.com
http://www.FGbeads.com