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Binding of Annexin to Phopholipids
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Binding of Annexin to Phopholipids

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Introduction Protocol References Credit lines
Category
Sugar binding proteins
Protocol Name

Binding of Annexin to Phopholipids

Authors
 Nakano, Yukiko
Graduate School of Humanities and Sciences, Ochanomizu University

Kojima-Aikawa, Kyoko *
Graduate School of Humanities and Sciences, Ochanomizu University
*To whom correspondence should be addressed.
KeyWords
Reagents
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[1. Co-precipitation test] Phospholipid: phosphatidylcholine, PC (Sigma-Aldrich, St. Louis, MO)

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[1. Co-precipitation test] Phospholipid: phophatidylserine, PS (Sigma-Aldrich)

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[2. Microtiter plate assay] Phophatidylserine, PS (Sigma-Aldrich)

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[2. Microtiter plate assay] Bovine serum albumin, BSA (Wako Pure Chemical Industries, Ltd., Osaka, Japan)

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[2. Microtiter plate assay] o-phenylenediamine, OPD (Wako Pure Chemical Industries, Ltd.)

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[2. Microtiter plate assay] Primary antibody: rabbit anti-GST polyclonal antibody (prepared in our Lab)

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[2. Microtiter plate assay] Secondary antibody: HRP-conjugated anti-rabbit IgG antibody (KPL, Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD)

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[2. Microtiter plate assay] Plastic micro titer plate (Immuron 1B, Thermo Fosher Scientific Inc., Waltham, MA)

Instruments
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[1. Co-precipitation test] Sonicator (Bransonic, Branson Ultrasonics Corporation, Danbury, CT)
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[1. Co-precipitation test] Slab gel electrophoresis apparatus (ATTO Corporation, Tokyo, Japan)
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[1. Co-precipitation test] Micro titer plate reader
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[1. Co-precipitation test] Block heater
Methods
1.

Co-precipitation test

1) 

 Suspend phospholipids (PC:PS = 1:1, 250 μg/mL) in TBS (Tris-buffered saline; 10 mM Tris-HCl pH 7.4, 150 mM NaCl) with a sonicator for 15 min to prepare phospholipid vesicles.

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2) 

 Take 20 μL of the above phospholipid solution and add 20 μL of annexin (100 μg/mL) in TBS containing 2 mM CaCl2.

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3) 

 Incubate the mixture at 37°C for 30 min with occasional mixing.

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4) 

 Centrifuge the mixture at 15,000 × g for 10 min, and then remove the supernatant.

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5) 

 Remove unbound annexin by washing twice. Add 40 μL of TBS containing 1 mM CaCl2 to the pellet, mix gently, and centrufuge at 15,000 × g for 10 min.

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6) 

 The pellet was subjected to SDS-gel electrophoresis by Laemmli’s method to detect annexin bound to phospholipids.

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2.

Microtiter plate assay (semi-quantitative ELISA method using recombinant GST (glutathione S-transferase) -fused annexin and anti-GST polyclonal antibody)

1) 

 Add 50 μL of methanol solution of PS (50–5,000 ng) to each well of a micro titer plate.

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2) 

 Bind PS to the bottom of the wells by warming the plate at 37°C on a block heater and evaporating off methanol. All other procedures were performed at room temperature.

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3) 

 Wash the plate three times with TBS (Tris-buffered saline; 10mM Tris-HCl, pH7.4, 150mM NaCl). The solutions and washes can be removed by flicking the plate over a suitable waste container or a sink.

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4) 

 Add 300 μL of 5% BSA/TBS and incubate for 1 h.

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5) 

 Add 100 μL GST-annexin (1 μg/mL) in TBS-Ca (5mM CaCl2/TBS) containing 2.5% BSA to each well and incubate for 1h.

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6) 

 Wash the plate three times with TBS-Ca.

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7) 

 Add 100 μL of the primary antibody (anti-GST polyclonal antibody) at a suitable dilution with 2.5% BSA/TBS-Ca containing to each well and incubate for 1 h.

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8) 

 Wash the plate three times with TBS-Ca.

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9) 

 Add 300 μL of 5% BSA/TBS-Ca and incubate for 20 min.

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10) 

 Add 100 μL of the secondary antibody (HRP-conjugated anti-rabbit IgG antibody) at a suitable dilution with 2.5% BSA/TBS-Ca and incubate for 30 min.

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11) 

 Wash the plate three times with TBS-Ca.

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12) 

 Add 150 μL of OPD solution [0.04% OPD, 0.08% H2O2/ 100mM Citrate-Phosphate buffer (pH5.0)] to each well. Incubate for 5–10 min. Positives appear orange color.

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13) 

 Add 50μL of 4M H2SO4 to each well to stop enzyme reaction.

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14) 

 Read the absorbance at 490 nm.

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Figure & Legends

Figure & Legends

Fig. 1. Binding of GST-human annexin 5 to phosphatidylserine

GST-human annexin 5 binding to phosphatidylserine was examined. Various amounts of phosphatidylserine were immobilized on a plastic plate and allowed to react with GST-human annexin5. Each point is the mean ± SD of three determinations.

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