Issue 5
January, 2015

Immunoprecipitation (1)Summary
FG beads® is suitable for various research fields including the chemical biology and also demonstrate superior performance inimmunoprecipitation.

Method for antibody immobilization on FG beads® :There are mainly two methods: one is to bind antibody covalently to beads by using activated functional groups such as NHS beads
beand
the other is to bind antibody to beads by Protein A, Protein G, etc.
Both beads have different advantages. NHS beads can reduce the amount of detached antibody in elution step and reduce non-specificnonbackground than use of Protein A and Protein G due to direct binding. On the other hand, Protein A and Protein G beads are easy
ea touse / bind the antibody with its strong affinity against IgGs.
We here show the advantages of our Protein G beads with higher recovery rate than other competitors by two different experiments.
experimResult (1)1. Experiment with the same amount of beads

2. Experiment to obtain the same recovery amounta of antigens

0.1mg of FG beads® have the capacity to bind antibody by 10ug or
more, and therefore can fully bind the antibody required for general
immunoprecipitation.

We performed immunoprecipitation to verify how much the beads ofeach company is required respectively to recover the same amountof antigens when 5ug of Anti-HSA is used.

We compared the recovery amounts of antigens for the beads(including FG beads®) of three companies, using 0.1mg of thebeads and 5ug of Anti-HSA.

The results show that the recovery amount of antigens for eachcompany becomes the same under the conditions shown below.

The results show that FG beads® have the largest recovery amount.

Competitor

Boil elution
(kDa)

MInFG

A

It is, therefore, confirmed that the amount of FG beads® required forthe immunoprecipitation is enough by 1/15 of that of competitor A
and 1/2.5 of that of competitor B.
Competitor

Boil elution
(kDa)

B

200
116
97.4

MInFG

A

B

200
116
97.4

66

HSA
Heavy chain

4566

HSA
Heavy chain

45

3131

Light chain

Light chain
21.5

21.5

14.4

14.4

6.5

6.5

Condition

Condition
Beads

Antibody

FG beads®

0.1mg

AB

Beads

Antibody

5ug

FG beads®

0.1mg

5ug

0.1mg

5ug

A1.5mg

5ug

0.1mg

5ug

B0.25mg

5ug※The above experiments were performed based on protocols
aarecommended by each company.

TAMAGAWA SEIKI CO., LTD.

http://www.FGbeads.com

Isolation of drug target protein
Materials
aterials and method
Mand method
FG beads® information■FG■Materials
1. Anti-HSA (Human Serum Albumin) (from Hytest)
2. HSA (from nacalai tesque)
3. HeLa cell extracts (cytosolic fraction) – 3mg/ml
4. Protein G beads
5. PBS(-) (137mM NaCl, 2.7mM KCl, 8.1mM Na2HPO4・12H2O,
1.5mM KH2PO4)
6. Wash Buffer A (10mM HEPES-NaOH(pH7.9), 50mM KCl,
0.2mM EDTA, 10%(v/v) glycerol)
7. Wash Buffer B (20mM HEPES-NaOH(pH7.9), 150mM KCl,
1mM MgCl2, 0.2mM CaCl2, 0.2mM EDTA, 10%(v/v) glycerol,
0.1% NP-40, 0.2mM PMSF)
8. Elution Buffer (0.1M glycine-HCl pH2.5)
9. Neutralization Buffer (1M Tris-HCl pH9.0)
10. SDS sample buffer (62.5mM Tris-HCl (pH6.8), 0.005% BPB,2% SDS, 10% glycerol, 5% 2-mercapto ethanol)

Product nameProtein G beads

Product number

TAS8848N1173

Storage temperature

2-8oC

Storage buffer

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

Size of beads

190nm ± 20nm

IgG binding capacity>100ug mouse IgG /mg of beads

Method 2 (Immunoprecipitation)(Immunoprecipitation■Method■Method 1 (Binding Antibody)
1. WashTransfer each amount of beads to a tube.
Wash beads with 200ul PBS(-) 2 times at 4oC.
2. Bind Antibody
Add antibody (5ug) diluted in 200ul PBS(-) to beads.
3. Reaction
Mix for 30min at room temperature.
4. Wash
Wash antibody binding beads with Wash buffer A
2 times at 4oC.

1. Add sample solution
Add 200ul HeLa cell extracts containing HSA to theantibody binding beads.
2. Reaction
Resuspend beads and incubate with rotation for 120min at 4oC.
3. Wash
Separate magnetically and remove supernatant.
Wash beads with Wash buffer B 3 times at 4oC.
Transfer 100ul resuspended solution to new tube to perform
2 elution pattern.
Separate magnetically and remove supernatant.
4. Elution
Acid elution
Add 28ul Elution buffer and resusupend beads.
Stand for 5min at 4oC, separate magnetically.
Transfer the supernatant to new clean tube and add 2ul
Neutralization buffer, and 10ul 4×SDS sample buffer.
Boil elution
Add 40ul 1×SDS sample buffer and resuspend beads.
Boil for 5min and remove the beads.
5. Analyze the samples by SDS-PAGE
SDS
and silver staining

Result (2)1. Experiment with the same amount of beads
Competitor

Acid elution
(kDa)

MInFG

A

2. Experiment to obtain the same recovery amounta of antigens
(kDa)

B

200

HSA
Heavy chain66

InFG ABHSA
Heavy chain

4545

31

M

200
116
97.4

116
97.4
66

Competitor

Acid elution

31

Light chain

Light chain

21.5

21.5

14.4

14.4

6.5

6.5

Acid elution samples show similar band pattern to Boil elution samples.

TAMAGAWA
SEIKI
CO.,
LTD.
TAMAGAWA
SEIKI
CO.,
LTD.

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