细胞核蛋白提取方法

Preparation of nuclear extracts from tissue

a. Dissect and mince 10-15 g of tissue. Adjust the volume of minced tissue to 30 ml with ice-cold tissue homogenization buffer. Homogenize in a tight-fitting Dounce homogenizer until >80-90% of the cells are broken as determined by microscopy.

b. To monitor lysis, mix 10 µl of the cell suspension with an equal volume of

0.4% Trypan Blue dye and examine the solution under a microscope equipped with a 20x objective. Lysed cells take up the dye and stain blue, whereas intact cells exclude dye and remain translucent. Continue to homogenize the tissue until >80-90% of the cells are broken.

c. Dilute the homogenate to 85 ml with ice-cold tissue homogenization buffer. Layer 27-ml aliquots over 10-ml cushions of ice-cold tissue homogenization buffer in ultraclear or polyallomer swinging-bucket centrifuge tubes. Centrifuge the tubes at 103,900g (24,000 rpm in a Beckman SW28 rotor) for 40 minutes at 4°C.

d. Decant the supernatant and allow the tubes to drain in an inverted position for 1-2 minutes. Place the tubes on ic

e. (Optional) Use a razor blade to cut off the top two thirds of the tube and place the bottom one third containing the nuclei on ice.

e. Resuspend the pellet of nuclei in 2 ml of ice-cold tissue resuspension buffer. Accurately measure the volume of the resuspended nuclei and add ice-cold 5 M NaCl to a final concentration of 300 mM. Mix the suspension gently. Incubate the suspension for 30 minutes on ice.

f. Recover the nuclei by centrifugation at 103,900g (24,000 rpm in a Beckman SW28 rotor) for 20 minutes at 4°C. Carefully transfer the supernatant to a fresh tube. Divide the supernatant into aliquots of 100-200 µl. Reserve an aliquot for protein concentration determination. Snap-freeze the remainder of the aliquots in liquid nitrogen, and store them in liquid nitrogen.

g. Determine the protein concentration of the supernatant by the Bradford method.

Cell homogenization buffer

10 mM HEPES-KOH (pH 7.9)

1.5 mM MgCl2

10 mM KCl

0.5 mM dithiothreitol

0.5 mM phenylmethylsulfonyl fluoride

PMSF

PMSF (Phenylmethylsulfonyl fluoride) C7H7FO2S or C6H5CH2SO2F is a highly toxic cholinesterase inhibitor. It is extremely destructive to the mucous membranes of the respiratory tract, eyes, and skin. It may be fatal by inhalation, ingestion, or skin absorption. Wear appropriate gloves and safety glasses. Always use in a chemical fume hood. In case of contact, immediately flush eyes or skin with copious amounts of water and discard contaminated clothing.

Cell resuspension buffer (17-1)

40 mM HEPES-KOH (pH 7.9)

0.4 M KCl

1 mM dithiothreitol

10% (v/v) glycerol

0.1 mM phenylmethylsulfonyl fluoride

0.1% (w/v) aprotinin

Store the buffer at 0°C until needed.

KCl

Dissolve an appropriate amount of solid KCl in H2O, autoclave for 20 minutes on liquid cycle and store at room temperature. Ideally, this 4 M solution should be divided into small (approx. 100 µl) aliquots in sterile tubes and each aliquot thereafter used one time.

10% (v/v) glycerol

To prepare a 10% (v/v) solution: Dilute 1 volume of molecular-biology-grade glycerol in 9 volumes of sterile pure H2O. Sterilize the solution by passing it through a prerinsed 0.22-µm filter. Store in 200-ml aliquots at 4°C.