troponin蛋白纯化 Protein purification: troponins

互联网2013-09-06

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Overview

TROPONINS

The calcium-dependent regulatory protein complex located on the thin actin filaments of muscle comprises of TnC (17.8 kDa), TnI (20.8 kDa), and TnT (30.5 kDa). These proteins are involved in the key regulatory mechanism for muscle contration.

Material

Source: skeletal muscle ether powder

Equipment:
• centrifuge
• Cibaron Blue-sephacryl column (5 x 50 cm)
• DEAE-sephadex A-50 column (2.5 x 25 cm)
• CM-sephadex C-50-120 column (2.5 x 25 cm)
• SDS-Page

Chemicals:
Tris-HCl, KCl, CaCl2, EDTA, DTT, 1 N HCl, 1 N KOH, imidazole, urea, citrate, NaN3

Have ready:
extraction solution: 1 M KCl, 25 mM Tris-HCl, 0.1 mM CaCl2, 0.1 mM DTT, pH 8.0

dialyzing buffer 1: 10 mM imidazole, 50 mM KCl, 0.1 mM CaCl2, 0.1 mM DTT, 0.02% NaN3, pH 7.0

dialyzing buffer 2: 2 mM Tris-HCl, 0.1 mM CaCl2, pH 8.0

dialyzing buffer 3: 6 M urea, 50 mM Tris-HCl, 1 mM EDTA, 0.1 mM DTT, pH 8.0

dialyzing buffer 4: 6 M urea, 50 mM citrate, 1 mM EDTA, 0.1 mM DTT, pH 6.0

Procedure

FLOW CHART

(all procedures are carried out at 4oC)

1. take skeletal muscle ether powder (~150 gram)

2. extract overnight with extraction solution (15:1, v/w); centrifuge at 10.9 x g for 10 min

3. (take pellet) reextract, add 1 M KCl to centrifuge bottles (7.5:1, v/w); centrifuge at 10.9 x g for 10 min

4. (take supernatant) lower pH to 4.6 by adding 1 N HCl, remove precipitate (tropomyosin) by centrifugation at 7020 x g for 10 min

5. (take supernatant) adjust pH to 8.0 by adding 1 N KOH; make 40% (NH4)2SO4 (230 gram/liter); centrifuge at 10.9 x g for 10 min

6. (take pellet) resuspend in minimal volume of dialyzing buffer 1; dialyze against 4 x 1 liter; clarify at 31,200 x g for 20 min

7. apply to blue sephacryl column

8. equilibrate with dialyzing buffer; wash with the same buffer (1 liter) and elute in steps of 0.1, 0.2, 0.3, 0.4, 0.5, and 0.6 M KCl, 1 liter each; collect 25 ml fractions >200/<220 ml; store frozen at -80oC or lyophilize at -20oC --> troponin 0.6 gram total

For TnC, TnT abd TnI:

1. (take pellet obtained in step 5) resuspend in minimal vol of dialyzing buffer 2; dialyze against 16 liter of dialyzing buffer 2; centrifuge at 31,300 x g for 15 min

2. (take supernatant) dialyze against dialyzing buffer 3 (2 x 2 liter)(urea settles down in the bag!)

3. apply to DEAE-sephadex A50 column

4. equilibrate with dialyzing buffer 3; elute with linear gradient 0-0.5 M KCl (2 x 500 ml); collect 6 ml fractions

To obtain pure TnI, TnC and TnT:
dialyze against dialyzing buffer 4 (1 liter) and load onto CM-sephadex column; elute with linear gradient 0-0.6 M NaCl (2 x 0.5 liter); collect 6 ml fractions-->TnI, TnC, TnT