Experiment Protocol 012
Competitive inhibition in screening by using ligand immobilized beads (Affinity
purification of proteins bind to ligands)
1. Materials
1.1 Ligand immobilized beads, Protein solution
・Ligand immobilized beads
Beads with different amount of immobilized ligands: 0.5 mg for each type
When investigating conditions other than the amount of ligand immobilization (e.g. protein
concentration, salt concentration of binding/washing buffer, etc.): 0.5 mg 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: 200 µL for each condition
・Ligands (for competitive inhibition)
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-40(NP-40) ・Dithiothreitol (DTT) ・Phenyl fluoride methane sulfonyl (PMSF)
・Dimethylsulfoxide ・Sample buffer (4×dye)
・Electrophoresis (SDS-PAGE) gel
・Electrophoresis buffer・Silver staining reagent
1.3 Apparatus
・Micro centrifuge ・Desktop centrifuge (for spin down)
・Magnetic stand (Tamagawa Seiki TA4899N12, etc.) ・Rotator
・Heat block ・Slab gel electrophoresis device
2. Method
2.1 Preparation for reagent solutions
1) 2×100 mM KCl buffer (500 mL): Mix 40 mL of 2.5 M KCl, 126 g of glycerol, 20 mL of 1 M
HEPES-NaOH solution (pH 7.9), 1 mL of 1 M MgCl2 solution, 200 µL of 1 M CaCl2 solution,
400 µL of 0.5 M EDTA solution(pH 8.0), and 10 mL of 10% NP-40 solution.Dilute this withultrapure water in a measuring cylinder to 500 mL total. (Store this buffer at room
temperature after filtration.)2) 100 mM KCl buffer: Mix 25 mL of ultrapure water and 25 mL of 2×100 mM KCl buffer. Add 50µL of 1 M DTT solution and 10 µL of 1 M PMSF solution just before use.
3) 1 M KCl buffer: Mix 18 mL of 2.5 M KCl, 7 mL of ultrapure water, and25 mL of 2×100 mM KClbuffer. Add 50 µL of 1 M DTT solution and 10 µL of 1 M PMSF solution just before use.
4) 1 M DTT solution: Prepare 1 M DTT solution by dissolving DTT in ultrapure water. (Store thisat -20℃)5) 1 M PMSF solution: Prepare 1 M PMSF solution by dissolving PMSF in dimethylsulfoxide.
(Store this at -20℃)
6)
20 mM ligand solution: Prepare 20 mM ligand solution by dissolving ligands indimethylsulfoxide.
7)
100 mM ligand solution: Prepare 100 mM ligand solution by dissolving ligands indimethylsulfoxide.

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Experiment Protocol 012
Composition of binding/washing buffer (100mM KCl buffer)
20 mM HEPES-NaOH(pH7.9), 100 mM KCl, 1 mM MgCl2, 0.2 mM CaCl2, 0.2 mM EDTA,10%(v/v) glycerol, 0.1% NP-40, 1 mM DTT, and 0.2 mM PMSF
Composition of 4×dye solution (Wako Pure Chemical Industries Ltd.：191-13272)
0.25 M Tris-HCl (pH 6.8), 0.02% BPB, 8% SDS, 40% glycerol, and 20% 2-mercaptoethanol
2.2 Procedures
1) Prepare 100 mM KCl buffer and 1 M KCl 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 15,000 rpm for 30 minutes or more
at 4℃ to remove insoluble matter. (Transfer the supernatant to a fresh tube after thecentrifugation.)
4)
Add ligand solution for competitive inhibition (Add only DMSO for the control withoutcompetitive inhibition) to the transferred protein solution until the concentration of DMSO
becomes 1%. In this case, the concentration of ligands will become 0 mM, 0.2mM, and 1 mM.
5) Preincubate for two hours at 4℃ by agitating with a rotator.
6)
During the preincubation, add 0.5 mg of ligand immobilized beads into each 1.5 mL
micro-tube. (Beads must be fully dispersed in advance to make a uniform suspension)7) Add 200 µL of 100 mM KCl buffer to the suspension and disperse the beads.
8) After spin down, separate magnetically, and discard the supernatant.
9) Repeat the above 7) to 8) two more times. (Wash the beads with buffer three times in total.)10)
Add 200 µL of preincubated protein solution to each 1.5 mL micro-tube containing beads
without the supernatant, and disperse the beads.
11) Perform binding reaction for four hours at 4℃ by agitating the beads with a rotator.
12) Four hours later, spin down, separate magnetically, and discard the supernatant.
13) Add 200 µL of 100 mM KCl buffer, and disperse the beads.
14) After spin down, separate magnetically, and discard the supernatant.
15) Repeat the above 13) to 14) two more times. (Wash the beads with buffer three times in total.)16) Add 30 µL of 1 M KCl buffer to the tube containing the beads without supernatant, anddisperse the beads.
17) Place the solution on the ice for five minutes to allow bound proteins to elute. After spin down,
separate magnetically.
18) Transfer the supernatant (salt elution sample) to a fresh 1.5 mL micro-tube.
19) Add 40 µL of 1×dye solution to the remaining beads, and disperse them.
20) Add 10 µL of 4×dye solution to the salt elution sample, and mix them.
21) Boil the beads dispersed solution and the salt elution sample for five minutes at 98℃. (using aheat block)22) Spin down the beads dispersed solution, and separate magnetically at room temperature.
23) Transfer the supernatant (the boil elution sample) to a fresh 1.5 mL micro-tube. (Discard thebeads.)
24) Proceed to the electrophoresis (SDS-PAGE) process. (Or store them in a freezer at -20℃.)
25) Apply the salt elution sample and the boil elution sample to SDS-PAGE. (e.g. 10 µL for each)26) Silver-stain the electrophoresed gel, and analyze it.

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Experiment Protocol 012
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 side
picture 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

After separation

Magnetic stand・ 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 (>109 Cells). Therefore, when conducting
the experiment on a small scale, the use of NP-40 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 awashing 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.

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Experiment Protocol 012
・The ligand concentration for competitive inhibition, such as 0.2 mM and 1 mM (the amount ofligands is 40 nmol and 200 nmol respectively) is set to 5 to 50 times of the estimated amount ofimmobilized ligands (4 nmol/0.5 mg). (The most suitable amount of immobilized ligands is generally
1 to 10 nmol per mg.)
Raise the concentration of ligands when you cannot confirm disappearance of a band, which iscaused by competitive inhibition, and which appears to be a band of target proteins.
If you cannot raise the concentration because the ligands are slightly soluble in DMSO, increase theamount of the ligands by raising the concentration of DMSO up to 5% when diluting them withbuffer.

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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