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Tandem affinity purification (TAP) is used to look at protein-protein interaction. Its use relies on generating a fusion protein with a TAP tag on the C- or N- terminal end. In this protocol, a two-step purification of N-terminus TAP-tagged proteins from yeast is described.

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[Bio101] TAP Purification of Yeast Proteins
[Bio101] 酵母蛋白的串联亲和纯化

生物化学 > 蛋白质 > 分离和纯化
作者: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
1/5/2011, 8411 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.17

[Abstract] Tandem affinity purification (TAP) is used to look at protein-protein interaction. Its use relies on generating a fusion protein with a TAP tag on the C- or N- terminal end. In this protocol, a two-step purification of N-terminus TAP-tagged proteins from yeast is described.

[Abstract] 酵母中N-端TAP-标记蛋白的二步纯化。

Materials and Reagents

  1. Complete Protease Inhibitor Cocktail Tablet (Roche Diagnostics)
  2. 100% NP-40 (Sigma-Aldrich)
  3. AcTEV Protease (Life Technologies, Invitrogen™)
  4. IgG Sepharose 6 Fast Flow (GE Healthcare Life Science)
  5. Calmodulin affinity resin (Guidechem/Stratagene)
  6. SDS lysis buffer
  7. Beta-ME
  8. Leupeptin
  9. Mg2 acetate
  10. Acetone
  11. BME
  12. EDTA
  13. CaCl2
  14. NaF
  15. Imidazole
  16. NP40 buffer (see Recipes)
  17. IPP150 buffer (see Recipes)
  18. IPP150 calmodulin binding buffer (see Recipes)
  19. TEV cleavage buffer (see Recipes)
  20. IPP150 calmodulin elution buffer (see Recipes)

Equipment

  1. Avestin Homogenizer (Avestin®)
  2. BECKMAN centrifuge and rotor (BECKMAN Coulter)
  3. Polycarbonate tubes
  4. Chromatography column
  5. Microfuge tube
  6. Shaker

Procedure

Day 1

  1. Grow 4,000 OD’s cells (two 2 L cultures in 8 L flasks, grow to 1 OD/ml) 30 °C shaker.
  2. Split to four 1 L centrifuge bottles. Spin at 7,000 rpm for 10 min. While spinning, make 60 ml NP40 Buffer before beginning. 
  3. Wash sample in each bottle with 250 ml cold H2O. Spin at 7,000 rpm for 10 min.
  4. Resuspend all cells in 50 ml total volume cold NP-40 buffer (add 30 ml buffer to first bottle, vortex, transfer all to next bottle, and so on).
  5. Lyse cells using Avestin Homogenizer (4 passes) – see instructions for usage before beginning.
  6. Split lysate into three polycarbonate tubes for 70 Ti Rotor for high-speed spin (~15 ml in each + 1 blank – weigh out to assure accuracy). 
  7. Spin at 100,000 x g for 30 min (37,500 rpm in an ultracentrifuge).
  8. Pour supernatants into a 50 ml conical. 
  9. Take 100 μl cleared lysate sample (100 μl of 50 ml sample = 1x).
  10. Prep IgG Sepharose in microfuge tube.
    a.  1 ml of IgG sepharose slurry (500 μl beads in 1:1 slurry) in 15 ml tube.
    b.  Wash 3x 5 NP-40 lysis buffer w/o PI’s 2 min 4,000 rpm.
  11. Add 1 ml cleared lysate directly to microfuge tube with beads and place beads into 50 ml conical with cleared lysate. Repeat once more with 1 ml more of lysate to obtain all of the beads.
  12. Rotate 2 h in cold room.
  13. Remove 1% of total sample (500 μl), wash 3x 14K for 30 sec with NP-40 buffer, resuspend in 100 μl SDS lysis buffer + 1x PI (IgG bound sample  – this is 5x).
  14. Pour samples over chromatography column in cold room (you will have to do this is stages because the column can only hold 10 ml at one time – keep samples cold while waiting).
    If samples are draining very slowly, it is because nucleic acids are plugging up column so you can always transfer everything to new column to speed things up.
  15. Take 100 μl IgG unbound sample from flowthrough (1x).    
  16. Wash column
    a.  3x 10 ml IPP150
    b.  1x 10 ml TEV Cleavage Buffer + DTT (after last wash, remove rest of TEV Buffer with a gel tip)
  17. Resuspend sepharose in 1 ml TEV Cleavage Buffer + DTT
    a.  Transfer to 1.5 ml Eppendorf.
    b.  Add 300 units TEV protease (30 μl– located in -80 freezer).
    c.  Rotate in cold room overnight

Day 2

  1. Spin down samples at 2,000 x g cold and place supernatant (1 ml) in 15 ml cold conical.
  2. Resuspend sepharose again in 1 ml TEV Cleavage Buffer + DTT.
    a.  Spin 2,000 x g cold, and add supernatant to same 15 ml conical (now you have a total of 2 ml).
  3. Spin 15 ml conical at 5,000 rpm for 5 min to pellet excess beads. Transfer supernatant to new cold 15 ml conical.
  4. Add 6 ml 0.1% Calmodulin binding buffer + BME + 6 μl 1 M CaCl2.
  5. Take 50 μl TEV cleaved sample (50 μl of 8 ml is 6.25x).
  6. To obtain TEV uncleaved sample
    a.   Add 1 ml TEV cleavage buffer to beads, spin 2,000 x g.
    b.  Wash 3x in TEV cleavage buffer and transfer to new tube on last wash.
    c.  Resuspend in 1ml TEV cleavage buffer (1.5 ml total).
    d.  Remove 45 μl of beads (3% of total).
    e.  Resuspended in 100 μl of SDS lysis buffer – 15x.
  7. Preparation of calmodulin beads – use 500 μl bead (1 ml of a 1:1 slurry).
    a.  Wash 3x 1 ml 0.1% calmodulin binding buffer 2 min 14 K in microfuge tube.
  8. Add beads to samples in 15 ml conical as in Step 8 of Day 1.
    a.  Bind samples in cold room 2 h.
  9. Pour samples over chromatography column in cold room.
  10. Take 100 μl calmodulin unbound sample (6.25x).
  11. Wash column
    a.  Wash 2x 1 ml 0.1% calmodulin binding buffer + BME.
    b.  Wash 1x 1 ml 0.02% calmodulin binding buffer + BME.
  12. Elute w/ 1 ml calmodulin elution buffer + BME (eluate 1) - collect in microfuge tube.
  13. Add 700 microliters calmodulin elution buffer + BME.
    a.  Keep in cold room for 10 min.
    b.  Eluate into Eppendorf (eluate 2).
  14. Take 20 μl of eluate 1 – 50x.
  15. Take 14 μl of eluate 2 – 50x.

TCA precipitate eluates

  1. Adjust eluates to 25% TCA w/ 100% TCA.
    a.  333 μl of TCA for 1 ml sample; 233 μl of TCA for 700 μl sample.
  2. Place samples on ice 30 min w/ periodic vortexing.
  3. Spin max cold for 10 min.
  4. Rotate 180 degrees, spin at max speed in the cold for 30 min (since pellet will collect away from center of centrifuge, you want to aspirate from side of eppendorf facing inwards).
  5. Wash 1x 1 ml cold acetone + 0.05 N HCl.
  6. Spin max cold 5 min, rotate 180 degrees spin max cold 5 min.
  7. Wash again w/ cold acetone.
  8. Spin max cold 5 min.
  9. Remove supernatant carefully, dry pellets.
  10. Freeze pellets -80 °C.

Recipes

  1. IPP150 buffer (100 ml)
    2.5 ml 1 M Tris-HCl (pH 8)
    25 mM Tris-HCl (pH 8)
    3 ml 5 M NaCl
    150 mM NaCl
    1 ml 10% NP-40
    0.1% NP-40
    Add H2O to final volume.


  2. TEV cleavage buffer (50 ml)
    0.5 M EDTA
    1.25 ml 1 M Tris-HCl (pH 8) 
    25 mM Tris-HCl (pH 8)
    1.5 ml 5 M NaCl 
    150 mM NaCl
    0.5 ml 10% NP-40
    0.1% NP-40
    50 ml 0.5 M EDTA
    Add H2O to final volume.


  3.  IPP150 calmodulin binding buffer (100 ml) – for 2x buffer without detergent
    2.5 ml 1 M Tris-HCl (pH 8) (25 mM)
    5 ml
    3 ml 5 M NaCl (150 mM)
    6 ml
    100 μl 1 M Mg2 Acetate (1 mM)
    200 μl
    100 μl 1 M Imidazole (1 mM)
    200 μl
    200 μl 1 M CaCl2 (2 mM)
    400 μl
    Add H2O to final volume.
    Divide into two 50 ml aliquots.
    Adjust one 50 ml aliquot to 0.1% NP-40 by adding 500 μl 10% NP-40.
    Adjust other 50 ml aliquot to 0.02% NP-40 by adding 100 μl 10% NP-40.
    Add 0.7 μl of 100% (m/v) beta-ME per ml before use (10 mM beta-ME).
  4. IPP150 calmodulin elution buffer (10 ml) – for 2x buffer without detergent
    0.25 ml 1 M Tris-HCl (pH 8) (25 mM)
    500 μl
    0.3 ml 5 M NaCl (150 mM)
    600 μl
    20 microliters 10% NP-40 (0.02%)
    40 μl
    10 microliters 0.5 M Mg2 Acetate (0.5 mM)
    20 μl
    10 microliters 1 M Imidazole (1 mM)
    20 μl
    400 microliters 0.5 M EGTA (20 mM)
    800 μl
    Add H2O to final volume.
    Add 0.7 μl of 100% (m/v) beta-ME per ml before use (10 mM beta-ME).

  5. NP-40 Buffer (1 L) – for 2x buffer without detergent
    1.61 g Na2HPO4*7H2O (6 mM)
    3.22 g
    0.553 g NaH2PO4*H2O (4 mM) 
    1.106 g
    100 ml 10% NP-40

    8.77 g NaCl (150 mM)
    17.54 g
    4 ml 0.5 M EDTA (4 mM)
    8 ml
    400 microliters leupeptin
    800 μl
    2.1 g NaF (50 mM)
    4.2 g
    0.0552 g Na3VO4 (300 μM)
    0.11 g
    Add H2O to final volume.
    Add the following protease inhibitors per 50 ml NP-40 buffer before use:
    1 complete tablet, EDTA free protease inhibitors (crush tablet first in weigh paper).
    100 microliters 0.5 M PMSF in DMSO.
  6. Samples to take to monitor efficiency
    Cleared lysate, IgG bound, IgG unbound, TEV cleaved, TEV uncleaved, calmoldulin unbound, eluate (pre-TCA precipitation).

Acknowledgments

This protocol has been modified and adapted in the Espenshade Lab, Johns Hopkins School of Medicine. Funding to support different projects that have used this protocol has come from NIH – National Heart, Lung, and Blood Institute, National Institute of Allergy and Infectious Diseases, the Pancreatic Cancer Action Network, and the American Heart Association.

References

  1. Rigaut, G., Shevchenko, A., Rutz, B., Wilm, M., Mann, M. and Seraphin, B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17(10): 1030-1032.

材料与试剂:

  1. 蛋白酶抑制剂Cocktail 片剂 (Roche)
  2. 100% NP-40 (Sigma)
  3. AcTEV蛋白酶 (Invitrogen)
  4. IgG Sepharose 6 Fast Flow (GE Healthcare)
  5. 钙调蛋白亲和树脂(Stratagene)

 

仪器:

  1. Avestin 匀浆机(Avestin)
  2. BECKMAN 离心机和转子 (BECKMAN)

 

步骤:

  1. 第一天

1)    生长到4,000 OD’s的细胞(28L的培养瓶各装2L的培养菌,长到1 OD/mL) 30°C摇菌。

2)    分到41L的离心瓶

7,000 rpm离心10 min

离心时,配60mls NP40 缓冲液(30mls 2X buffer + 6mls 10% NP40 + 24 mls 冰的dH20冰育)

每个瓶子加250ml冷水洗样品

 7,000 rpm离心10 min

3)    50 mL NP-40 缓冲液(加30 mL 缓冲液到第一个瓶中,震荡,转移到另一个瓶等等)重悬样品。

4)    使用Avestin匀浆机裂解细胞使用之前参考Espenshade lab提供的说明书。

5)    将裂解物分装到三个聚丙烯管,70 Ti高速离心(~15 mls /+ 1 空白保证称量精准)。100,000g离心30min37,500rpm)。

6)    上清倒入50ml锥形瓶。

100 ul 澄清的裂解样品(50 ml样品的100 ul = 1X

7)    准备IgG Sepharose

--15ml管中加入1ml IgG sepharose 悬浮液(1:1悬浮液500 ul 磁珠)

 --3x5 NP-40裂解缓冲液洗,4K rpm 离心2 mins

8)    直接加1ml澄清的裂解液到有磁珠的管子中,将带有磁珠的裂解液加到50ml的三角瓶中,重复一次再加1ml到管子中,确保所有的珠子都被转移到锥形瓶中。

9)    低温旋转孵育2h

10)1%样品 (500ul) NP-40 Buffer3x 14K 30s,加100 ul SDS裂解缓冲液重悬 + 1x PI IgG结合样品这里是5倍)。

11)低温将样品倒入层析柱(只能这样做,因为层析柱一次只能容纳10 mls–等待时保持低温)。如果样品排出很慢,可能是核酸堵塞柱子,你可以将这一柱子中的所有东西转移到一个新柱子中来加快流速。

12)流出物(1x)中取出100 ul IgG非结合样品。

13)洗柱子

-3x 10 mL IPP150

-1x 10 mL TEV裂解液+DTT (最后洗净后,用枪头除去TEV缓冲液)

14)1 mL TEV裂解液+ DTT重悬珠子

-转移到1.5 mL离心管中

-300单位 TEV蛋白酶 (30 ul –-80℃储存)

-过夜低温旋转孵育

  1. 第二天

1)    2,000 g低温离心,取1ml上清到15 mL冷圆锥瓶。

2)    1 mL TEV裂解液+ DTT再重悬Sepharose

- 2,000 g 低温离心 1’, 将上清液加到同一 15 mL 锥形瓶中 (现在总体积为2 mL)

3)    16.  5,000rpm离心15ml锥形瓶5 min 来沉淀剩余的柱子.  将所有的转移到新的15ml 锥形瓶中.

4)    加入6 mL 0.1% 钙调蛋白结合缓冲液+ BME + 6 ul 1M CaCl2

5)    50 ul TEV裂解样品(50 ul of 8mls is 6.25X)

6)    获得 TEV未裂解样品:

- 1 mL TEV 到珠子里2,000 g 离心1’,

- TEV Cleavage Buffer 洗三次,最后一次转移到新的管子中。

- 1ml TEV Cleavage Buffer重悬 (1.5 ml total)

- 移除 45 ul 珠子(总量的3% ),

- 100 ul SDS裂解buf重悬 – 15X

7)    准备钙调蛋白的珠子 500 ul 磁珠(1mL of a 1:1 slurry)

- 洗涤3次,用1 mL 0.1% 钙调蛋白结合缓冲液 14000rpm 离心 2min

8)    和第一天的第八步一样,加磁珠到15ml锥形瓶中。. 

-在低温下结合样品两个小时

9)    低温下,加样品到层析柱。

10)100 ul钙调非结合蛋白(6.25X)

11)洗脱柱子

-2x 1 mL 0.1% 钙调蛋白结合缓冲液+ BME

-1x 1 mL 0.02%钙调蛋白结合缓冲液+ BME

12)洗脱液 w/ 1 mL钙调蛋白洗脱缓冲液 + BME (洗脱液1)–收集到离心管中

13)700 mL钙调蛋白洗脱缓冲液+ BME

- 低温保持 10’

-洗脱到离心管子中 (洗脱物 2)

14)20 ul洗脱液1 – 50X

14 ul洗脱液2 – 50X

  1. TCA沉淀洗脱液

1)    配洗脱液25% TCA w/ 100% TCA 

1ml样品加333 ul TCA700 ul 233 ul TCA

将样品放置冰上,30’ w/定期震荡

2)    低温最大转速离心10’

3)  180 度旋转, 最大速离心30’ (如果沉淀偏移管子的中心,你需要从相对着沉淀的管子的一面吸取。)

4)    1x 1mL 冷丙酮 + 0.05N HCl

5)    低温最大速度离心 5’, 180 度旋转最大速度低温离心 5’

6)    用冷的丙酮再洗涤一次

7)    最大速度低温离心 5’

8)    除去上清,干燥沉淀

9)    -80冷冻沉淀

 

方法:

  1. IPP150 缓冲液(100 mL)

2.5 mL 1M Tris-HCl pH 8                     25mM Tris-HCl pH 8

3 mL 5M NaCl                                        150mM NaCl

1 mL 10% NP-40                                      0.1% NP-40

加水到最终体积

  1. TEV 裂解缓冲液 (50 mL)

1.25 mL 1M Tris-HCl pH 8                     25mM Tris-HCl pH 8

1.5 mL 5M NaCl                                       150mM NaCl

0.5 mL 10% NP-40                                     0.1% NP-40

50 mL 0.5M EDTA                                      0.5mM EDTA

加水到最终体积

使用之前加0.5 ul 1M DTT/mL    (0.5mM DTT)        

  1. IPP150 钙调蛋白结合缓冲液(100 mL) – 无洗涤剂 2X缓冲液

2.5 mL 1M Tris-HCl pH 8 (25mM)       5 mL

3 mL 5M NaCl    (150mM)               6 mL

100 ul1M 醋酸镁 (1mM)              200 ul

100 ul 1M咪唑  (1mM)              200 ul

200 ul 1M CaCl2 (2mM)              400 ul

加水到最终体积

分成两个50 mL

一个加入500mL 10% NP-40配成0.1% NP-40

另一个加入100mL 10% NP-40配成0.02% NP-40

使用前每毫升加入0.7 ul 100% (m/v) beta-ME (10mM beta-ME)

  1. IPP150 钙调蛋白洗脱缓冲液(10 mL) – 无洗涤剂 2X缓冲液

0.25 mL 1M Tris-HCl pH 8  (25mM)        500 ul

0.3 mL 5M NaCl     (150mM)               600 ul

20 mL 10% NP-40 (0.02%)                  40 ul

10 mL 0.5M 醋酸镁 (0.5mM)               20 ul

10 mL 1M Imidazole (1mM)               20 ul

400 mL 0.5M EGTA (20mM)               800 ul

加水到最终体积

使用前每毫升加 0.7 ul 100% (m/v) beta-ME (10mM beta-ME)

  1. NP-40缓冲液 (1L) – 无洗涤剂 2X缓冲液

1.61 g Na2HPO4*7H2O     (6mM)         3.22 g           

0.553 g NaH2PO4*H2O (4mM)         1.106 g

100 mL 10% NP-40                

8.77 g NaCl        (150mM)       17.54 g

4 mL 0.5 M EDTA    (4mM)         8 ml

400mL亮肽素           800 ul

2.1 g NaF          (50mM)        4.2 g

0.0552 g Na3VO4     (300uM)       0.11g

加水到最终体积

使用前每50毫升NP-40缓冲液加下面蛋白酶抑制剂:

一个片剂,EDTA蛋白酶抑制剂(称重之前粉碎片剂),每100mL DMSO  0.5M PMSF

有效的检测样品:

裂解液,结合IgG抗体,非结合IgGTEV裂解,TEV非裂解,非结合的钙调蛋白,洗脱液(TCA沉淀前)

 

参考文献

1.        Rigaut G., Shevchenko A., Rutz B., Wilm M., Mann M., Seraphin B. (1999). A generic protein purification method for protein complex characterization and proteome exploration. Nature Biotechnology 17(10): 1030-2. 

2.        Puig O., Caspary F., Rigaut G., Rutz B., Bouveret E., Bragado-Nilsson E., Wilm M., Seraphin B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24(3): 218-29. 

 

 

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How to cite this protocol: Tong, Z. (2011). TAP Purification of Yeast Proteins. Bio-protocol Bio101: e17. DOI: 10.21769/BioProtoc.17; Full Text



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