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Galveston, Texas
Biomolecular Resource Facility
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Galveston, Texas 77555-0144
 
   
   
   

Nuclear and Cytoplasmic Extracts

 

Materials:

 

• Endonuclease (Sigma E8263)

• 10% Triton X or IGEPAL 630

• Phosphatase inhibitor Solution

• Phosphate buffered Saline (PBS), pH 7.4

• Solution A (see recipe at the end of protocol)

• Solution B (see recipe at the end of protocol)

• Solution C (see recipe at the end of protocol)

• DeStreak Rehydration Solution (Amersham-Biosciences)

 

Method:

 

1. Wash cell plate with ice-cold PBS. Add 1ml ice-cold PBS with Phosphatase inhibitor

solution per 10cm plate and scrape cells. Transfer to an ice-cold Eppendorf tube.

 

2. Pellet cells at 1000 X g for 2 minutes. Remove supernatant.

 

3. Add Solution A without resuspending the pellet at a 1:1 (v/v) ratio to that of the

cellular pellet. Centrifuge as in step 2. Remove supernatant.

 

4. Add Solution A at a 1.5:1 volume of Solution A per volume of cell pellet. Briefly vortex

or resuspend pellet, incubate on ice for 10 minutes. Add 1/20th volume [cell pellet +

Solution A] of 10% Triton X (or IGEPAL 630) to a final concentration of 0.5% and mix.

Centrifuge 6000 rpm @ 4 ºC for 30 seconds.

 

5. Collect supernatant (cytoplasmic extract). Determine concentration, aliquot into 200 μg samples and store immediately at –80 ºC.

 

6. Add Solution B in a 1:1 volume relative to nuclear pellet and gently resuspend.

Centrifuge 12,000 rpm @ 4 ºC for 10 minutes. Discard supernatant.

 

7. Add DeStreak Rehydration solution in a 2:1 ratio to the pellet. Vortex for 5

minutes at room temperature.

 

8. Add endonuclease (Sigma, E8263) to a final concentration of 300 units/ml and

incubate at room temperature for 30 minutes.

 

9. Sonicate the sample for 10 seconds. Centrifuge 13k rpm @ 4 ºC for 30 minutes.

Collect supernatant (nuclear extract). Determine protein concentration.

 

10. Aliquot into 200 μg samples and store immediately at –80 ºC.

 

Reagents and Solutions:

 

A. Solution A (low salt buffer)

 

Components

Stock conc.

Working conc.

Vol. from stock

HEPES, pH 7.9

1.0 M

50 mM

1 ml

KCl

2.0 M

10 mM

100 μl

EDTA pH 8.0

0.2 M

1 mM

100 μl

EGTA pH 8.0

0.2 M

1 mM

100 μl

DTT *

1.0 M

1 mM

20 μl

PMSF *

0.1 M

0.5 mM

100 μl

Pepstatin A

5 μg/μl

1 μg/ml

4 μl

Leupeptin

5 μg/μl

1 μg/ml

4 μl

STI

10 μg/μl

10 μg/ml

20 μl

Aprotinin

10 μg/μl

10 μg/ml

20 μl

Na3VO4

0.1 M

1 mM

200 μl

Na4P2O7

0.1 M

1mM

200 μl

NaF

0.5 M

20mM

800 μl

Note:

*DTT and PMSF should be added immediately prior to use

  1. Add M.Q. H2O to a final volume of 20 ml.
  2. First add DTT then PMSF and quickly vortex vigorously
  3. Keep the solution on ice and store at 4°C

 

B. Solution B (low salt sucrose buffer)

 

Components

Stock conc.

Working conc.

Vol. from stock

HEPES pH 7.9

1.0 M

50 mM

1 ml

KCl

2.0 M

10 mM

100 μl

EDTA pH 8.0

0.2 M

1 mM

100 μl

EGTA pH 8.0

0.2 M

1 mM

100 μl

DTT *

1.0 M

1 mM

20 μl

PMSF *

0.1 M

0.5 mM

100 μl

Pepstatin A

5 μg/μl

1 μg/ml

4 μl

Leupeptin

5 μg/μl

1 μg/ml

4 μl

STI

10 μg/μl

10 μg/ml

20 μl

Aprotinin

10 μg/μl

10 μg/ml

20 μl

Na3VO4

0.1 M

1 mM

200 μl

Na4P2O7

0.1 M

1 mM

200 μl

NaF

0.5 M

20 mM

800 μl

Note:

*DTT and PMSF should be added immediately prior to use

  1. Add 6.8 grams sucrose (final concentration 1.0 M)
  2. Add M.Q. H2O to a final volume of 20 ml.
  3. First add DTT then PMSF and quickly vortex vigorously
  4. Keep the solution on ice and store at 4°C

 

C. Solution C (High salt buffer)

 

Components

Stock conc.

Working conc.

Vol. from stock

HEPES pH 7.9

1.0 M

50 mM

1 ml

KCl

2.0 M

400 mM

4 ml

EDTA pH 8.0

0.2 M

1 mM

100 μl

EGTA pH 8.0

 0.2 M

 1 mM

 100 μl

DTT *

 1.0M

 1 mM

 20 μl

PMSF *

 0.1 M

 0.5 mM

 100 μl

Pepstatin A

 5 μg/μl

 1 μg/ml

 4 μl

Leupeptin

 5 μg/μl

 1 μg/ml

 4 μl

STI

 10 μg/μl

 10 μg/ml

 20 μl

Aprotinin

 10 μg/μl

 10 μg/ml

 20 μl

NaF

 0.5 M

 20 mM

 800 μl

Na3VO4

 0.1 M

 1 mM

 200 μl

Na4P2O7

 0.1 M

 1 mM

 200 μl

Glycerol

 100%

 10%

 2 ml

Note:

*DTT and PMSF should be added immediately prior to use

  1. Add M.Q. H2O to a final volume of 2 0ml
  2. Add DTT first then PMSF and quickly vortex vigorously
  3. Keep the solution on ice and store buffer at 4°C

 

D. Phosphatase Inhibitor Salt Solution

 

50 X Preparation. Dilute to 1 X in Phosphate Buffered Saline (1X PBS)

 

Component

50 X concentration

Working Conc.

Amount for 50ml of 50X

NaF

 1 M

 20 mM

 2.1 grams

Na4P2O7•10 H2O

 0.05 M

 1 mM

 1.12 grams

Na3VO4

 0.05 M

 1 mM

 0.46 grams

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