Issue 6April, 2015

Immunoprecipitation (2)SummaryImmnoprecipitation is an effective approach for the detection of a slightamount of endogenous protein in cells. This time, we performed
immunoprecipitation of p16 of HeLa cell, by using Protein G beads.
p16 is known as one of inhibitors of cyclin-dependent
dependent kinase (CDK), andis also referred to as p16 INK4a because it belongs to INK4 family. The
cell cycle is composed as shown in the right figure. CDK4/6 is activated byforming a complex with Cyclin D, phosphorylates target protein, andinduces progress in the cell cycle from G1 phase to S phase. p16 works tostop the progress of the cell cycle by competitively inhibiting the complex
formation of CDK4/6 and Cyclin D.
It is known that p16 gene functions as a tumor suppressor gene, and ismutated or deleted in a human cancer cell in most cases. When a cell
receives oncogenic stress or signals, an expression of p16 gene increases,
the function of CDK4/6 is inhibited, and the cell cycle progression isstopped . This is regarded as a self-defense
defense mechanism to protect normal
cell from canceration.

Inhibition pattern of cell cycle progression by p16

ResultUsing 0.1mg of Protein G beads from each company and 5ug of Anti-p16
Antiantibody, we immunoprecipitated p16 from HeLa cell extracts,
and performed silver staining and Western blotting after SDS-PAGE.
PAGE.
As the result of the Western blotting, we comfirmed that the p16 was purified and FG beads® achieved the largest recovery amount.
The result of the silver staining showed that the proteins interacting with the p16 were co-immunoprecipitatedcobecause plural clear bandswere detected.
Competitor(kDa)

M

FGA

B

Competitor(kDa)

InM

FGA

B

250
150
100
75

250
150
10075
Heavy chain

50

Heavy chain

503737

Light chain

2520

Light chain

2520

p16
1515

1010
Silver staining

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Western blotting

http://www.FGbeads.com

Materials
aterials and method
Mand method■Materials
1. Protein G beads
2. HeLa cell extracts (cytosolic fraction) – 3mg/ml
3. PBS(-) (137mM NaCl, 2.7mM KCl, 8.1mM Na2HPO4・12H2O,1.5mM KH2PO4)
4. Wash Buffer A (10mM HEPES-NaOH(pH7.9), 50mM KCl,
0.2mM EDTA, 10%(v/v) glycerol)
5. 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)
6. SDS sample buffer (62.5mM Tris-HCl (pH6.8), 0.005% BPB, 2% SDS,10% glycerol, 5% 2-mercapto ethanol)
7. Anti-p16 antibody (from Abcam)
8. Anti-mouse IgG, HRP-Linked Whole Ab Sheep(from GE healthcare)
9. Transfer buffer (25mM Tris, 192mM Glycine, 20%(v/v) Methanol)
10. Blocking buffer (from Thermo)
11. TBS-T buffer (20mM Tris-HCl (pH7.5), 500mM NaCl, 0.1% Tween20)■Method 1 (Binding Antibody)
1. WashTransfer 0.1mg 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.

■Method 2 (Immunoprecipitation)

beads® information■■FGProduct nameProtein G beadsProduct number

TAS8848N1173

Storage temperature

2-8oC

Storage buffer

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

Size of beads

190nm ± 20 nm

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

3 (Western blotting)■■Methods
1. Perform SDS-PAGE, and place the gel in Transfer buffer for
about 10min.
2. Transfer the protein from the gel to a PVDF membrane.
3. Block the membrane with the Blocking buffer for 15min at room
temperature.
4. Dilute the primary antibody with the Blocking buffer to 1/150.
5. Incubate for 60min at room temperature.
6. Wash the membrane with TBS-T buffer 3 times.
7. Dilute the secondary antibody with the TBS-T buffer to 1/2000.
8. Incubate for 60min at room temperature.
9. Wash the membrane with TBS-T buffer 5 times.
10. Detect with a chemiluminescence substrate.

1. Add sample solution
Add 200ul HeLa cell extracts .
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.
Separate magnetically and remove supernatant.
4. Elution
Add 40ul SDS sample buffer and resuspend beads.
Boil for 5min and remove the beads.
5. Analyze the samples by SDS-PAGE and silver staining

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

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