Dynabeads®His�Tag�Isolation�and�Pulldowny g

2010/2�����

Overview

• Dynabeads His-Tag Isolation and Pulldownphysical / magnetic properties� physical / magnetic properties

• Application data for protein isolations� Workflow� ReproducibilityReproducibility� Stability� Competitor study

• Summary

2

Dynabeads His-tag Isolation and Pulldown

• Uniform monosized (1μm diameter)• Uniform, monosized (1μm diameter) superparamagnetic beads

• Beads coated with novel IMAC h i f h l i C 2

2µm

chemistry for chelating Co2+

• Same high specificity of TALON™ chemistry but with greatly enhanced y g ycapacity

• Protein binding occurs on the bead surface onlysurface only

� Fast interaction� No target size limit� Low backgroundg� Easy handling� Secondary applications

• Ideal for automation

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• Ideal for automation

TALON™ is a registered trademark of Clontech Laboratories Inc., USA.

His-Tag Isolation Protocol

Magnetic Separation

Bind Wash Elute

Secondary Applications!y pp

4

From lysate to purified protein in 20 min!

Testing Dynabeads for His-tag binding

EGFPfluorescence

Bioanalyser(SDS-PAGE)

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GFP Fluorescence Measure vs Bioanalyser readoutreadout

GFP FluorescenceGFP readouty = 52.48x - 267.67

3 0E+04

4.0E+04

5.0E+04

6.0E+04

nce

GFP (ng/well) Fluorescence

1000 52363

500 25784

0.0E+00

1.0E+04

2.0E+04

3.0E+04

0 100 200 300 400 500 600 700 800 900 1000 1100

Fluo

resc

en

250 12659

125 6224

62.5 3013

0 30 5-1.0E+04

0 100 200 300 400 500 600 700 800 900 1000 1100

Quantity (ng)

A B

0 30.5

Isol

ate

A

Isol

ate

B

Ladd

er

kDa

Bioanalyser readout

Isolate A Isolate B

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GFP Binding Capacity and Bioanalyser DataData

GFP Binding Capacity

50

60

Bioanalyser Results of Selected Batches

800

900140

ProteinPurity

20

30

40

50

µg G

FP/m

g be

ads

300

400

500

600

700

Prot

ein/

µl E

luat

e

40

60

80

100

120

% P

urity

0

10

RD1 RD2 1 2 3 4 5 6 7 8 9 10 11 12 13

µ

0

100

200

RD1 8 9 10 13

ng

0

20

40

• GFP binding capacity vs Bioanalyzer quantification of Selected Dynabeads• GFP binding capacity vs Bioanalyzer quantification of Selected Dynabeads batches

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Accelerated Stability Study of 2 Different Dynabead batches120

140

LK152 4C

LK152 RTA

Dynabead batches

40

60

80

100

% o

f 4C

LK152 RT

LK152 37C

LK439 4C

LK439 RT

LK439 37C

A

ABB

B

0

20

0 5 10 15 20 25 30 35 40 45

Days

100

120

140

1604C

LK152 4C

LK152 RT

LK152 37C

LK439 4C

AA

AB

20

40

60

80

% o

f LK439 4C

LK439 RT

LK439 37C

B

B

B

07 16 22 42

Days

• Aliquots of batches A and B were stored at 4oC, RT, and 37oC for up to 42 d

8

days.

• Purity is >90% for all (data not shown)

Competitor Comparison: Bead Morphology

• Compared different magnetic ‘beads’ by light microscopy

Dynabeads

microscopy

• 40x enhancement for all

P B Q

M N C

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Competitor Comparison: Capacity Per Equal Volume of BeadsEqual Volume of Beads

Competitor Comparison900 120

Protein

Purity

500

600

700

800

n/µl

Elu

ate

60

80

100

Pur

ity

ng/µl StDev Purity StDev

Dynabeads 547.5 0.7 99.2 1.1

P 621.0 144.3 37.1 0.5

100

200

300

400

ng P

rote

in

20

40

%P B 244.3 161.0 79.6 23.1

Q 263.0 43.8 93.7 8.9

M 88.5 5.0 100.0 0.0

N 119.0 49.5 52.5 2.8

0Dynabeads P B Q M N C

0 C 405.5 176.1 85.9 5.9Dynabeads P B Q M N C

• The amount and purity of the protein isolated with 50μl of each bead slurry were compared

• Competitor P, B, and N have short incubations (5 min; most similar to our protocol)

• Competitor Q, M, and C have very long incubations (30-60 min)

• Best competitors � Q and C� Similar purity and decent capacity � The others lack in either purity or capacity or both

HOWEVER:

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• HOWEVER:� Capacity per μl bead is ~57% of Dynabeads® His-Tag Isolation and Pulldown (GFP quantification results; data

not shown)� Isolation time from lysate to purified protein takes well over one hour.

SummarySummary

• Uniform monosized (1 μm) superparamagnetic beads bind protein on bead surface

� Fast interactionN t t i li it� No target size limit

� Secondary applications� Reproducibility

Easy handling� Easy handling

• Highly stable

C tit t d h• Competitor study show:� Highly competitive capacity per μl (list price)

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