Reggie Stallworth
S1
Myosin S1 Prep. Using Papain Digest
Solutions:
A. Ammonia Dialysis Buffer
0.2 M Ammonium Acetate (pH 7.2)
0.002 M EDTA
0.002 M MgCl2 (if S1 is to be prepared)
D. S1 Purification Buffer A
0.05 M Tris-HCl (pH 8.0)
0.001M DTT
0.002M MgCl2 (or .002 M EDTA)
B. Papain Digest Solution
Stock papain diluted to [0.5-1.0 mg/ml] in:
0.005M cysteine (pH6.0)
0.002M EDTA
E. S1 Purification Buffer B
0.5 M KCl
C. Stop Digestion Solution
0.1 M iodoacetic acid
F. Dialysis Buffer
0.1M KCl
0.01M DTT
0.0003 M EGTA
0.01M Imidazole (pH 7.0)
IMPORTANT REMINDERS:
Keep protein solution on ice and buffers cold at ALL times!
Use ONLY cold H2O for solutions made throughout the prep!
PROCEDURE:
(SKIP DEGLYCERINATE MYOSIN TO S1 PREP IF MYOSIN IS NOT GLYCERINATED)
Deglycerinate Myosin
pH
7.0
Myosin Buffer
(MB)
Myosin Precipitate
Buffer (MPB)
7.0
Chemical
KCL
EDTA
MOPS
[Stock]
0.5M
1.0M
1x
Volume/L
37.28g
0.2 ml
5 ml
EDTA
MOPS
0.5M
1.0M
4 ml
20 ml
6x
Volume/L
223.68g
1.2ml
30ml
[Final]
500mM
0.1mM
5mM
2mM
20mM
1. Remove x ml of amount of glycerinated myosin from –20oC freezer.
a. [concentration of myosin]
2
b.
x ml
y
=y
=ml of myosin
2 Add glycerinated myosin to myosin precipitate buffer (MSB); fill to 1L with MSB.
Allow to myosin to precipitate on ice for approx. 20 minutes. Centrifuge until all myosin
is pelleted.
3. Add 6x Myosin Buffer (MB) to dissolve pellet. Immediately add ddH20 to bring
solution to 1mg/ml.
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Reggie Stallworth
S1
4. Repeat steps 2-3 adding myosin solution to approx. .5L. Repeat twice to remove all
glycerin from myosin.
5. Read [ ]; adjust to 20mg/ml with 1xMB.
S1 Prep
1.Dialyze [myosin] 2% against Ammonia Dialysis Buffer overnight. (To ensure
complete
precipitation dialyzate is changed at least once- Total Volume of
dialyzate 2L).
2. Bring myosin suspension to room temperature.
3.Digestion is started by addition of Papain Digest Solution to a final papain [ ] of
.03mg/ml
4. Rxn is run for 7 minutes.
5. Stop rxn by addition of iodoacetate acid (Stop Digestion Solution) to a final [ ] of
.001M.
6. Suspension is run for 90 minutes at 31,000rpms in 70 Ti. (27000 Beckman 30 rotor) to
remove all insoluble digestion products.
7.Yield of S1 in supernatant is ~ 40% of the theo value.
Mg. S1 is enriched in DTNB light chain-------EDTA. S1 is deficient in that light chain
Further purification of S1
Using ion-exchange chromatography
8. Solution of S1 (400-500mg) at 7-10 mg/ml is dialyzed against S1 Purification Buffer A
and applied to a 2.5 x 60 cm column of DE-52 equilibrated in the same buffer.
9.Wash column with ~350ml and retained S1 is eluted with a linear KCl gradient. (S1
Purification Buffer B)
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S1
S1 Purification DE-52 Column
0.6
0.5
0.4
Abs
0.3
0.2
0.1
-0.1
1
61
121
181
241
301
361
421
481
541
601
661
721
781
841
901
961
1021
1081
1141
1201
1261
1321
1381
1441
1501
1561
1621
1681
1741
1801
1861
1921
1981
2041
2101
2161
2221
0
-0.2
Time(secx10)
10. Collect and pool the fractions from the front 2/3 of the protein peak in 6-7 sections..
Collect 50 l of each section to run gel and determine the most concentrated section.
Myosin
std
1464sec 1518sec
to
to
1512sec 1584sec
1590sec
to
1656sec
Myosin
std
1662sec
to
1728sec
1734sec
to
1800sec
(sec x 10)
11. Further purify with ammonium sulfate fractionation.
12. Material precipitated at 47% is discarded.
13. Protein fraction salting out between 47-58% saturation is collected (Sorvall SS34
rotor, 15000 rpms, 20 minutes)
14.Protein is resuspended in a minimum amount of solution G.
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S1
15. Exhaustive dialysis against this buffer is required to ensure removal of traces of
ammonium sulfate.
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