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Nuclear Extracts

This document provides instructions for preparing nuclear extracts from HeLa cells for in vitro transcription and footprinting experiments. It describes buffers and procedures for lysing cells, isolating nuclei, and extracting nuclear proteins. Nuclei are resuspended and homogenized in buffers, then subjected to centrifugation to separate nuclear and cytoplasmic fractions. The nuclear pellet is resuspended and dialyzed to produce a nuclear extract, while the supernatant is further centrifuged and dialyzed to yield an S100 cytoplasmic extract. Expected yields are 6-8 mg/ml for the nuclear extract.

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65 views2 pages

Nuclear Extracts

This document provides instructions for preparing nuclear extracts from HeLa cells for in vitro transcription and footprinting experiments. It describes buffers and procedures for lysing cells, isolating nuclei, and extracting nuclear proteins. Nuclei are resuspended and homogenized in buffers, then subjected to centrifugation to separate nuclear and cytoplasmic fractions. The nuclear pellet is resuspended and dialyzed to produce a nuclear extract, while the supernatant is further centrifuged and dialyzed to yield an S100 cytoplasmic extract. Expected yields are 6-8 mg/ml for the nuclear extract.

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biotech_vidhya
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© © All Rights Reserved
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PREPARATION OF NUCLEAR EXTRACTS

FOR IN VITRO TRANSCRIPTIONS, FOOTPRINTING, ETC.

All buffers, autoclave then add HEPES or filter sterilize

Nuclei extract buffers: Dignam et al., Nuc. Acid Res. 11, 1475-1489

A: 10 mM HEPES (pH7.9 at 4°C) 1 ml 1M


1.5 mM MgCl2 150 µl 1M
10 mM KCl 500 µl 2M
[Add fresh 0.5mM DTT]
TVf = 100mls

B: 0.3 M HEPES 7.9 30 mls 1M


1.4 M KCL 70 mls 2M
0.03 M MgCl2 3 mls 1M
TVf = 100 mls

C: 20 mM HEPES 7.9 2 mls 1M


25% v/v glycerol 50 mls 50%
0.42 M NaCl 10.5 mls. 4M
1.5 mM MgCl2 150 µl 1M
0.2 mM EDTA 40 µl 0.5 M
[Add PMSF to 0.5mM fresh]
[0.5 mM DTT fresh]
TVf = 100mls

D: 20 mM HEPES 7.9 2 mls 1M


20% v/v glycerol 40 mls 50%
0.1 M KCL 5 mls 2M
0.2 mM EDTA 40 µl 0.5 M
[0.5 mM DTT]
[Add DMSF to 0.5 mM fresh]
TVf = 100 mls

Stocks:100x DTT = 50 mM or 200x = 100 mM = 15.4 mg/1 ml


100x PMSF = 50 mM 200x = 100 mM = 77 mg/ml
1M HEPES 7.9 = 23.8 g/100mls 0.01 M = .238 g/100mls
4 M KCL = 29.82 g/100mls
50% glycerol need 100mls
1 M MgCl2 = 20.33 g/100mls
PBS

III.C.1
(FOR HELA CELLS)

TEN MAXI PLATES--~2-2.5 x 108 HeLa cells


Pellet ~2.5mls

Buffer: A - 20 mls
C - 1 ml
D - 200 mls
B - 1 ml

1. Wash cells w/PBS, ice cold.


2. Scrape in 5 mls PBS/Plate.
3. Pellet at 2000 RPM 10' clinical centrifuge.
4. Resuspend in 5 x vol. Buffer A, 4°C, ice 10' ~12mls.
5. Pellet at 2000 RPM 10', 4°C.
6. Resuspend in 2 x vol. Buffer A ~5mls.
7. Homogenize ~20 strokes.
8. Check lysis on microscope.
9. Pellet at 2000 RPM 10',4°C.

From this point, treat the pellet and supernatant separately.

A. Pellet = nuclei
1. Transfer to ultracentrifuge tubes.
2. Pellet at 25,000g 4°C = 17,000 RPM.
3. Resuspend in 0.6 mls buffer C.
4. Homogenize, Vf~1.2-1.5 ml.
5. Stir w/magnet 30' at 4°C.
6. Pellet at 25,000g, 30'.
7. Dialysis SUPER (~1ml) against buffer D.
for 5 hours
8. Pellet at 25,000g, 20'
9. SUPER = nuclear extract
freeze

expect 6-8 mgs/ml

B. Super = cytoplasmic extract


1. Add to Super 0.11 vol. buffer B (e.g., 6 mls + 0.66 ml buffer B)
2. Spin 100,000 g, 1 hr. at 4°C.
3. Dialysis of Supernatent (~4-5 mls) against buffer D (200 mls) for 5-8 hours.
4. This is S100 fraction. Freeze

III.C.2

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