Experiment Protocol m11
Immunoprecipitation by using antibody immobilized beads
1. Materials
1.1 Antibody Immobilized beads, Protein solution
・Antibody immobilized beads
Antibody (-) beads and antibody (+) beads: m.1 mg for each type
When investigating conditions (e.g. protein concentration, salt concentration of binding /washing buffer, etc.): m.1mg for each condition
・Protein solution
Protein concentration: 5 to 15 mg/mL (Not applicable if the original concentration is less than
5 mg/mL.)
Dilute the solution with binding/washing buffer (usually protein concentration should be 1mg/mL). Required volume : 2mm µl for each condition
1.2 Reagents・2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)
・Sodium hydroxide (NaOH) ・Potassium chloride (KCl) ・Magnesium chloride (MgCl2)
・Calcium chloride (CaCl2) ・Ethylenediamine tetraacetic acid (EDTA) ・Glycerol (Glycerin)・Nonidet P-4m (NP-4m)
・Phenyl fluoride methane sulfonyl (PMSF)
・Dimethylsulfoxide ・Sample buffer (4×dye）
・Glycine ・Tris (hydroxymethyl) amino methane (Tris) ・Hydrochloric acid (HCl)・Electrophoresis (SDS-PAGE) gel ・Electrophoresis buffer ・Silver staining reagent
1.3 Apparatus
・Micro high-speed cold centrifuge ・Desktop centrifuge (for spin down)
・Magnetic stand (Tamagawa Seiki TA4899N1, etc.) ・Rotator
・Heat block ・Slab gel electrophoresis device
2. Method
2.1 Preparation for reagent solutions
1) 2×15m mM KCl buffer (5mm mL): Mix 6m mL of 2.5 M KCl, 126 g of glycerol, 2m mL of 1 M
HEPES-NaOH solution (pH 7.9), 1 mL of 1 M MgCl2 solution, 2mm µL of 1 M CaCl2 solution, 4mmµL of m.5 M EDTA solution(pH 8.m), and 1m mL of 1m% NP-4m solution.Dilute this with ultrapure
water in a measuring cylinder to 5mm mL total. (Store this at room temperature after filtration.)2) 15m mM KCl buffer: Mix 25 mL of ultrapure water and 25 mL of 2×15m mM KCl buffer. 1m µL of
1 M PMSF solution just before use.
3) 1 M PMSF solution: Prepare 1 M PMSF solution by dissolving PMSF in dimethylsulfoxide. (Storethis at -2m℃)
Composition of binding/washing buffer (15mmM KCl buffer)
2m mM HEPES-NaOH(pH7.9), 15m mM KCl, 1 mM MgCl2, m.2 mM CaCl2, m.2 mM EDTA,
1m %( v/v) glycerol, m.1% NP-4m, and m.2 mM PMSF

Composition of acid elution buffer
m.1 M Glycine (pH 2.5)

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Experiment Protocol m11
Composition of neutralizing buffer
1 M Tris-HCl (pH 9.m)
Composition of 4×dye solution (Wako Pure Chemical Industries Ltd.: 191-13272)
m.25 M Tris-HCl(pH 6.8), m.m2% BPB, 8% SDS, 4m% glycerol, and 2m% 2-mercaptoethanol
2.2 Procedures
2.2.1 Preparation
1) Prepare 15m mM KCl buffer, 1 M KCl buffer and acid elution buffer, and place them on ice.
2) On the ice, adjust the concentration of the protein solution to a target concentration (1 mg/mL,3 mg/mL, etc.)
3) Add the solution into 1.5 mL micro-tubes, and centrifuge at 15,mmm rpm for 3m minutes or more
at 4℃ to remove insoluble matter. (Transfer the supernatant to a fresh tube after thecentrifugation.)4) During the centrifugation, add m.1 mg of antibody immobilized beads into each 1.5 mL
micro-tube. (Beads must be fully dispersed in advance to make a uniform suspension)5) Add 2mm µL of 15m mM KCl buffer to the suspension and disperse the beads.
6) After spin down, separate magnetically, and discard the supernatant.
7) Repeat the above 5) to 6) two more times. (Wash the beads with buffer three times in total.)
2.2.2 Binding and washing1) Add 2mm µL of protein solution obtained after the centrifugation to each 1.5 mL micro-tube
containing beads without the supernatant, and disperse the beads.
2) Perform binding reaction for two hours at 4℃ by agitating the beads with a rotator.
3) Two hours later, spin down, separate magnetically, and discard the supernatant.
4) Add 2mm µl of 15m mM KCl buffer, and disperse the beads.
5) After spin down, separate magnetically, and discard the supernatant.
6) Repeat the above 3) to 4) two more times. (Wash the beads with buffer three times in total.)
2.2.3 Elution
2.2.3.1 In case of acid elution
1) Add 28 µL of acid elution buffer to the beads without supernatant, and disperse the beads.
2) Place the solution on the ice for five minutes to elute acid. After spin down, separatemagnetically.
3) Transfer the supernatant (acid elution sample) to a fresh 1.5 mL micro-tube. (Discard thebeads.)
4) Add 2µL of neutralizing buffer to the acid elution sample to neutralize the sample.
5) Add 1m µL of 4×dye solution to the sample, and mix them.
6) Boil them for five minutes at 4℃. (using a heat block)

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Experiment Protocol m11
2.2.3.2 In case of boil elution
1) Add 4m µL of 1×dye solution to the beads without supernatant, and disperse the beads.
2) Boil them for five minutes at 98℃. (using a heat block)3) Spin down the beads dispersed solution, and separate magnetically at room temperature.
4) Transfer the supernatant (the boil elution sample) to a fresh 1.5 mL micro-tube. (Discardthe beads.)
2.2.4 Analysis of proteins
1) Proceed to the electrophoresis (SDS-PAGE) process. (Or store them in a freezer at -2m℃.)
2) Apply the acid elution sample and the boil elution sample to SDS-PAGE. (e.g. 1m µl for each)3) Silver-stain the electrophoresed gel, and analyze it.
3. Supplements
・Beads are easily dispersed by the manual agitation. In the manual dispersion method, the bottom ofa micro-tube is glided over an uneven surface (side of plastic test tube rack in this case) creating
turbulence through the collisions. (see left side picture below)
Please make sure to use well-constructed tubes with the caps tightly secured in order to prevent
leakage/breakage. Use of cap lock is recommended in order to prevent leakage. (see right sidepicture below).
For more information, please visit FG beads web site and see the movie of the method.
(Please click: http://www.magneticnanoparticle.jp/en/htdocs/technique/affinity.html for movingpictures.)・Perform magnetic separation by placing a magnetic stand on ice.

Before separation

Magnetic stand3

After separation

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Experiment Protocol m11
・ For protein solution, we recommend using plasmatic compartments (or nuclear fractions ormembrane fractions) prepared by Dignam method.
A reference book：J.D.Dignam, R.M.Lebovitz, and R.G.Roeder, Nucleic Acids Res. 11, 1475(1983)
The Dignam method, however, requires numerous cells (>1m9 Cells). Therefore, when conducting
the experiment on a small scale, the use of NP-4m lysis method or marketed cell extract reagentsare also allowable.
・The salt elution is used to isolate weak affinity proteins. When the affinity is disregarded because alltypes of proteins can be eluted by boil elution, the salt elution should be excluded. Conversely,
when you need to recover strong affinity proteins only, strong affinity proteins alone can be purified
by increasing the volume of salt elution buffer, and increasing the number of the elution in a
washing process.
・Salt concentration of the salt elution sample is so high that it may yield a white turbidity orprecipitation. And as it does not affect the results of the experiment, proceed directly to theelectrophoresis.
4. Notes
・Be sure to centrifuge the cell extract before mixing with beads. Otherwise, insoluble fractions
caused by freezing and thawing remain in the extract, which can cause a background.
・Recover beads not by centrifugation but by magnetic separation. If centrifuged, insoluble fractionsof proteins yielded during the reaction are recovered along with the beads, which can cause abackground.
・When dispersing beads in washing and elution process, ensure that the beads are fully dispersed.
Otherwise, non-specific bands are likely to appear.
・Wear gloves during the experiment to prevent keratin contamination.

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