Enabling Epigenetics Research

Nuclear Extract Kit
Catalog Nos. 40010 & 40410(Version D7)

Copyright 2022 Active Motif, Inc.

Revision

Date

Description of Change

D7

March, 2024

Important: Phosphatase Inhibitors (AM Part 102146 ) has been
replaced with 100x Phosphatase Inhibitors (AM Part 105840).
Consult the appropriate protocol (nuclear, cytoplasmic or whole-cell
extract from cells or from tissue) to determine the updated amountof PBS/Phosphatase Inhibitors solution that will be required foryour extraction(s).

D4December, 2018

Clarified recommendations for measuring protein concentration.

The Nuclear Extract Kit is for research use only. Not for use in diagnostic procedures.
Information in this manual is subject to change without notice and does not constitute a
commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any
warranty of any kind, either explicit or implied. Information may be changed or updated inthis manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated,
in whole or in part, without the prior written consent of Active Motif, Inc. This documentation
is proprietary information and protected by the copyright laws of the United States andinternational treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2022 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All
rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to theirrespective companies.

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Contents
Overview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Performance and Benefits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  2
Kit Components and Storage  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Optional Additional Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  4
Protocols – Buffer Preparation and Recommendations . . . . . . . . . . . . . . . . . . . . . . .  5
Reagents from the Nuclear Extract Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Protocol I – Adherent or Suspension Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
A. Preparation of Nuclear Extract from Cells  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .  7
B. Preparation of Whole-Cell Extract from Cells  . . . . . . . . . . . . . . . . . . . . . . . . . . . .  9
Protocol II – Fresh or Frozen Tissue  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
A. Preparation of Nuclear Extract from Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
B. Preparation of Whole-Cell Extract from Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Section A. Example Dilution for Bradford Protein Quantitation . . . . . . . . . . . . . . . . 14
Section B. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Technical Services . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

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Overview
The Nuclear Extract Kit has been developed for the preparation of nuclear, whole-cell andcytoplasmic extracts from cells or tissue. The kit procedure is a simple, fast and effective way
to obtain non-denatured, active proteins contained in cytoplasmic and nuclear compartmentsof the cell. The proteins collected by this high-salt extraction method can be used for avariety of standard protocols including electrophoretic mobility shift assay (EMSA), DNAfootprinting, Western blotting and preparative purification of nuclear proteins for use indownstream applications such as enzyme activity assays or binding studies. The Nuclear
Extract Kit is the recommended method for preparation of cell extracts for use in Active
Motif’s TransAM™ assays to monitor transcription factor activation.
Each kit provides reagents for 100 or 400 extractions from 8.8 x 106 cells, which corresponds
to HeLa cells grown to confluence in a 100 mm tissue culture dish.
The Nuclear Extract Kit is intended for use for preparation of nuclear, cytoplasmic and wholecell fractions from various cell and tissue types. Extractions can be performed with fresh andfrozen cell and tissue samples.
For preparation of nuclear extracts, the cells are first collected in ice-cold PBS in thepresence of Phosphatase Inhibitors to limit further protein modifications (expression,
proteolysis, dephosphorylation, etc.). Then, the cells are resuspended in Hypotonic Buffer
to swell the cell membrane and make it fragile. Addition of the Detergent causes leakage ofthe cytoplasmic proteins into the supernatant. After collection of the cytoplasmic fraction, thenuclei are lysed and the nuclear proteins are solubilized in detergent-free Lysis Buffer in thepresence of the Protease Inhibitor Cocktail.
To prepare whole-cell extracts, cells are collected in the PBS/Phosphatase Inhibitors
solution and lysed in the Lysis Buffer. Solubilized proteins are separated from the cell debris
by centrifugation.
To prepare extracts from fresh or frozen tissue, samples are diced and disrupted using a
Dounce homogenizer to form a single-cell slurry. Cells are then lysed in the lysis buffer andsolubilized proteins are separated from the cell debris by centrifugation.
The protein concentration of the cell extract is then measured by Active Motif’s ProStain™
Protein Quantification Kit (Catalog No. 15001) or a Bradford-based assay. We do notrecommend the BCA assay as the appropriate dilution required will produce such a dilute
extract, the protein concentration will not be within the range of the standard curve
produced.
Product
Nuclear Extract Kit

FormatCatalog Nos.

100 rxns

40010

400 rxns

40410

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Kit Performance and Benefits
Assay Time

2 hours

Yield of protein

Cytoplasmic extract: ~0.5-1 mg at ~1-2 mg/mL from 8.8 x 106 cells
Nuclear extract: ~0.15-0.25 mg at ~3-5 mg/mL from 8.8 x 106 cells
Whole-cell extract ~1.2-2.4 mg at 4-8 mg/mL from 8.8 x 106 cells
Note: Example is from HeLa cells grown to confluency

Assay compatibility

TransAM™, EMSA, DNA footprinting, Western blotting, preparative nuclear
protein purification, protein assays. Active transcription factors extracted include
NFκB, AP-1, CREB, p53, HIF-1, STAT, Sp1, NFAT, MyoD, NF-YA, C/EBP and PPARγ.
Successful extraction has been performed with HeLa, WI-38, COS-7, PC-12,
Jurkat and RINm5F cells, and many more. Extraction can be performed withfresh or frozen cell and tissue samples.

Time Course Studies

For time course studies where the effectors are added at different timepoints,
we suggest that cells be lysed and processed at the same time to maintainconsistency among samples.

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Figure 1: Specific extraction of nuclear and cytoplasmic extracts.
NFκB activation was assayed with the TransAM® NFκB p50 Kit (Catalog Nos. 41096 and 41596)
using increasing amounts of nuclear and cytoplasmic extracts isolated from unstimulated andstimulated HeLa cells using the Nuclear Extract Kit.

Cy

Nu

WC

NP
a-Tubulin
COX IVGAPDH
Lamin A/C
Pan-Cadherin
RNA pol II

Figure 2: Western blots of specific proteins from cellular fractions.
Active Motif’s Nuclear Extract Kit was used to generate nuclear, cytoplasmic and whole-cell
extracts from HeLa cells to analyze the efficiency of cellular fractionation. 20 μg of cytoplasmic(Cy), nuclear (Nu), and whole-cell (WC) extracts, along with the nuclear pellet (NP) collected from
nuclear extraction, were run on 4-20% SDS-PAGE gels and analyzed by Western blot using 1:1000
dilutions of primary antibodies to various specific proteins. Primary antibodies: Active Motif alphaTubulin mAb (Catalog No. 39528); COX IV mAb; GAPDH mAb; Active Motif Lamin A/C mAb (Catalog
No. 39288); Pan-Cadherin mAb; Active Motif RNA pol II mAb (Catalog No. 39097). Note that RNA
pol II is found primarily in the nuclear pellet due to its association with the nucleoskeleton andchromatin. The optional addition of Detergent to the nuclear pellet can sometimes aid in solubilizing
nucleoskeleton- or chromatin-associated proteins such as RNA pol II.

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3Kit Components and Storage
Kit components can be stored at -20ºC prior to first use. Then, we recommend storing eachcomponent at the temperatures recommended in the table below:
Kit Component

Qty.
100 rxns

Qty.
400 rxns

Storage

Lysis Buffer AM1

10 mL

50 mL

4°C for 6 months

1 M Dithiothreitol (DTT)

100 µL500 µL-20°C for 1 year

Protease Inhibitor Cocktail

100 µL500 µL-20°C for 1 year

10X PBS100 µL4 x 100 mL

4°C for 6 months

100x Phosphatase Inhibitors

8 mL

4 x 8 mL

-20°C for6 months

10X Hypotonic Buffer

50 mL

4 x 50 mL

4°C for 6 months

Detergent

3 mL

4 x 3 mL

4°C for 1 year

Additional Materials Required• 5 and 10 mL pipettes• Pipettors• Cell scraper• 15 mL conical tubes• Microcentrifuge tubes• Centrifuge (with swinging buckets adapted to 15 mL conical tubes) and microcentrifuge
pre-cooled at 4ºC• Rocking platform• Distilled water

Optional Additional Materials
Dounce homogenizer, 2 mL capacity, with a large (looser, A) pestle clearance of 0.12 mm
for the initial sample reduction, and a small (tighter, B) pestle clearance of 0.06 mm for the
final homogenate
Note:

Adherent and suspension cell preparations require the small-clearance (B)pestle whereas fresh and frozen tissue preparations require both large (A)and small (B)-clearance pestles.
Suggested Dounce homogenizer: Active Motif (Catalog Nos. 40401 and 40415)

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Protocols – Buffer Preparation and Recommendations
Reagents from the Nuclear Extract Kit
Preparation of PBS/Phosphatase Inhibitors
Imporant: As of May, 2022, there is a new formulation of Phosphatase Inhibitors. The
originial Phosphatase Inhibitors (AM Part 102146), have been replaced by 100x Phosphatase
Inhibiors (AM Part 105840). Consult the appropriate protocol (nuclear, cytoplasmic or wholecell extract from cells or from tissue) to determine the updated amount of PBS/Phosphatase
Inhibitors solution that will be required for your extraction(s).
For example, to make nuclear extract from a 100 mm plate of cells, prepare 8 mL of PBS/
Phosphatase Inhibitors solution as follows: mix 0.8 mL 10X PBS in 7.12 mL distilled water,then add 80 µL Phosphatase Inhibitors. Since the Phosphatase Inhibitors lose their activity
24 hours after dilution, the solution should be used within this time frame. Any remaining
solution should be discarded if not used on the same day.
Preparation of 1X Hypotonic Buffer
Note that the Hypotonic Buffer already contains phosphatase (but not protease) inhibitors.
Consult the protocol you intend to use (nuclear, cytoplasmic or whole-cell extract from cells
or from tissue) to determine the amount of 1X Hypotonic Buffer that will be required for yourextraction(s). For example, to make nuclear extract from a 100 mm plate of cells, prepare 500µL of 1X Hypotonic Buffer as follows: mix 50 µL 10X Hypotonic Buffer and 450 µL distilledwater. Any remaining 1X Hypotonic Buffer can be stored at 4ºC for 1 week.
Preparation of 10 mM DTT
The Nuclear Extract Kit is supplied with 1 M DTT. To make Complete Lysis Buffer, you willneed to first dilute the 1 M DTT to create 10 mM DTT. However, 1 M DTT is used to perform
nuclear and cytoplasmic extractions from tissue (see Protocol II: Fresh or Frozen Tissue on
page 11). To make 10 mM DTT, make a 1:100 dilution of the supplied 1 M DTT in distilled water.
For example, add 1 µL of 1 M DTT to 99 µL distilled water. DTT is highly labile, so 10 mM DTT
must be prepared fresh each time. Avoid multiple freeze/thaw cycles.

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5Preparation of Complete Lysis BufferNote:

If you will be using the nuclear extract in a TransAM Kit that comes with Lysis
Buffer AM2, substitute the Lysis Buffer AM2 in for Lysis AM1 when preparing
the Complete Lysis Buffer.

Note that the Complete Lysis Buffer already contains phosphatase (but not protease)inhibitors. The presence of phosphatase inhibitors gives a yellow coloration to Lysis Buffer
AM1. Consult the appropriate protocol (nuclear, cytoplasmic or whole-cell extract from cells orfrom tissue) to determine the amount of Complete Lysis Buffer that will be required for yourextraction(s). For example, to make nuclear extract from a 100 mm plate of cells, prepare 50µL Complete Lysis Buffer as follows: add 5 µL 10 mM DTT to 44.5 µL of Lysis Buffer AM1,
then add 0.5 µL Protease Inhibitor Cocktail. Since some of the protease inhibitors lose theiractivity promptly after dilution. Therefore, use the Complete Lysis Buffer immediately for cell
lysis. Any remaining amount should be discarded if not used on the same day.

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Protocol I – Adherent or Suspension Cells
A. Preparation of Nuclear Extract from Cells
The following protocol is based on samples of approximately 8.8 x 106 cells, whichcorrespond to HeLa cells grown to confluence in a 100 mm tissue culture plate. Each sample
is one reaction. Prepare PBS/Phosphatase Inhibitors, Hypotonic Buffer and Complete Lysis
Buffer as described above on page 5 in the section Buffer Preparation. Adjust the volumes
according to the buffer preparation chart if using plates of different sizes. Place buffers andany tubes needed on ice before beginning assay.
Quick Chart for Preparing Buffers for Nuclear Extraction from Cells
60mm plate or 100mm plate or
3.2 x 106 cells
8.8 x 106 cells

150mm plateor 2 x 107 cells

Reagents to prepare

Components

PBS/Phosphatase
Inhibitors

10X PBS

0.4 mL

0.8 mL

1.6 m

Distilled water

3.56 mL7.12 mL

14.24 mL

Phosphatase Inhibitors

40 µL

80 µL

160 µL

Total Required

4.0 mL

8.0 mL

16.0 mL

10X Hypotonic Buffer

25 µL

50.0 µL

100 µL

Distilled water

225 µL450.0 µL

0.9 mL

Total Required

250 µL500 µL

1 mL

10 mM DTT

2.5 µL

5 µL

10.0 µL

Lysis Buffer AM1**

22.25 µL

44.5 µL

89 µL

Protease Inhibitor Cocktail

0.25 µL

0.5 µL1.0 µL

Total Required

25 µL

50 µL

100 µL

Total Required

1.25 µL

2.5 µL

5 µL

1X Hypotonic Buffer

Complete Lysis Buffer*(Optional) Detergent*The addition of Detergent to the nuclear pellet may help with solubility of proteins, specificallythose tightly associated with membranes or chromatin.
**If the nuclear extract will be used in a TransAM Kit that comes with Lysis Buffer AM2, substitute the
Lysis Buffer AM2 in for Lysis Buffer AM2 here.

Step 1: Cell Collection
1.

Aspirate media out of dish. Wash with 5 mL ice-cold PBS/Phosphatase Inhibitors.
Aspirate solution out and add 3 mL ice-cold PBS/Phosphatase Inhibitors. If working
with suspension cells, pellet cells and wash with 3 mL ice-cold PBS/Phosphatase
Inhibitors and proceed to step 3.

2.

Remove cells from dish by gently scraping adherent cells with cell lifter or,
alternatively, pellet suspension cells by centrifugation. Transfer cells to a pre-chilled
15 mL conical tube.

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Note:

Use of trypsin to detach cells can activate some signal transduction pathways.

3.

Centrifuge cell suspension for 5 minutes at 200 x g in a centrifuge pre-cooled at 4ºC.

4.

Discard supernatant. Keep cell pellet on ice.

Step 2: Cytoplasmic Fraction Collection
Note:

Make sure to verify cell lysis efficiency and release of the nuclei by comparing
the appearance of cells before and after cell lysis (steps 1 and 2) using phasecontrast microscopy. Intact cells should appear as a dark central nucleus
surrounded by a halo of less dense cytoplasm.

1.

Gently resuspend cells in 500 µL 1X Hypotonic Buffer by pipetting up and down
several times. Transfer to a pre-chilled microcentrifuge tube. Allow cells to swell by
incubating for 15 minutes on ice.

2.

Add 25 µL Detergent (for tissue, add 5 µL Detergent per every 100 µL Hypotonic
Buffer) and vortex 10 seconds at the highest setting.

3.

Check a small sample under the microscope to verify that cells have been efficiently
lysed and that nuclei have been released. If the cells are not adequately lysed at thisstep, use an ice-cold Dounce homogenizer with a small-clearance (B) pestle to lyse
the cells (refer to the Troubleshooting Guide in Section A of the Appendix, page 14,
for further information).

Note:

Do not proceed with the centrifugation step until the cells are
sufficiently lysed.

4.

Centrifuge suspension for 30 seconds at 14,000 x g in a microcentrifuge pre-cooled
at 4ºC.

5.

Transfer supernatant (cytoplasmic fraction) into a pre-chilled microcentrifuge
tube. Store the supernatant at -80ºC until ready to use. Use the pellet for nuclear
fraction collection.
Optional: Save a fraction of the supernatant (cytoplasmic fraction) for determinationof fractionation efficiency by Western blot.

Step 3: Nuclear Fraction Collection
1.

Resuspend nuclear pellet in 50 µL Complete Lysis Buffer by pipetting up and down.
Optional: Add 2.5 µL Detergent to help solubilize membrane-associatednuclear proteins.
A very viscous pellet may form and not completely resuspend. Alternatively, a Dounce
homogenizer can be used (refer to Appendix, Section B Troubleshooting Guide on
page 15, for further information). Vortex 10 seconds at the highest setting.

2.

Incubate suspension for 30 minutes on ice on a rocking platform set at 150 rpm.

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3.

Vortex 30 seconds at the highest setting. Centrifuge for 10 minutes at 14,000 x g ina microcentrifuge pre-cooled at 4ºC. Transfer supernatant (nuclear fraction) into a
pre-chilled microcentrifuge tube. Optional: Save a fraction of the supernatant (nuclear
fraction) for determination of fractionation efficiency by Western blot.

4.

Aliquot and store at -80ºC. Avoid freeze/thaw cycles.

Note:

The presence of certain detergents or PMSF may interfere with the Bradford
assay, thus perform a 1:50 or 1:250 dilution of your samples. Use theComplete Lysis Buffer as the blank with a similar dilution as the samples.
Read the absorbance at 595 nm with a plate reader spectrophotometer(refer to the Appendix, Section A on page 14 for an example dilution series).
As an alternative, try Active Motif’s ProStain™ Protein Quantification Kit(Catalog No. 15001), which offers greater sensitivity and resistance to many
contaminating agents.

B. Preparation of Whole-Cell Extract from Cells
The following protocol is based on samples of approximately 8.8 x 106 cells, whichcorrespond to HeLa cells grown to confluence in a 100 mm tissue culture plate. Each sample
is one reaction. Prepare PBS/Phosphatase Inhibitors and Complete Lysis Buffer as describedabove on page 5 in the section Buffer Preparation. Adjust the volumes according to thebuffer preparation chart if using plates of different sizes. Place buffers and any tubes neededon ice before beginning assay.
Quick Chart for Preparing Buffers for Whole-Cell Extraction from Cells
60mm plate or 100mm plate or
3.2 x 106 cells
8.8 x 106 cells

150mm plateor 2 x 107 cells

Reagents to prepare

Components

PBS/Phosphatase
Inhibitors

10X PBS

0.4 mL

0.8 mL

1.6 m

Distilled water

3.56 mL7.12 mL

14.24 mL

Phosphatase Inhibitors

40 µL

80 µL

160 µL

Total Required

4.0 mL

8.0 mL

16.0 mL

10 mM DTT

2.5 µL

5 µL

10.0 µL

Lysis Buffer AM1

22.25 µL

44.5 µL

89 µL

Protease Inhibitor Cocktail

0.25 µL

0.5 µL1.0 µL

25 µL

50 µL

100 µL

Complete Lysis Buffer

Total Required

Step 1: Cell Collection
1.

Aspirate media out of dish. Wash with 5 mL ice-cold PBS/Phosphatase Inhibitors.
Aspirate solution out, and add 3 mL ice-cold PBS/Phosphatase Inhibitors. If working
with suspension cells, pellet cells and wash with 3 mL ice-cold PBS/Phosphatase
Inhibitors and proceed to step 3.

2.

Remove cells from dish by gently scraping with cell lifter. Transfer cells to a pre-chilled
15 mL conical tube.
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3.

Centrifuge cell suspension for 5 minutes at 200 x g in a centrifuge pre-cooled at 4ºC.

4.

Discard supernatant. Keep cell pellet on ice.

Step 2: Cell Lysis
1.

Resuspend cell pellet in 300 µL Complete Lysis Buffer by pipetting up and down.

2.

Incubate suspension for 10-30 minutes on ice on a rocking platform set at 150 rpm.
Check a small sample under the microscope to verify that cells have been efficiently
lysed to determine length of incubation. If the cells are not adequately lysed at thisstep, use an ice-cold Dounce homogenizer with a small-clearance (B) pestle to lyse
the cells (refer to Appendix, Section A Troubleshooting Guide on page 15, forfurther information).

Note:

Do not proceed with the centrifugation step until the cells are
sufficiently lysed.

3.

Vortex 30 seconds at the highest setting. Centrifuge for 20 minutes at 14,000 x g in a
microcentrifuge pre-cooled at 4ºC. Transfer supernatant (whole-cell extract) into a prechilled microcentrifuge tube.

4.

Aliquot and store at -80ºC. Avoid freeze/thaw cycles.
Optional: Save a fraction of the supernatant for determination of extraction efficiency
by Western blot.

Note:

The presence of certain detergents or PMSF may interfere with the Bradford
assay, thus perform a 1:50 or 1:250 dilution of your samples. Use theComplete Lysis Buffer as the blank with a similar dilution as the samples.
Read the absorbance at 595 nm with a plate reader spectrophotometer(refer to the Appendix, Section A on page 14 for an example dilution series).
As an alternative, try Active Motif’s ProStain™ Protein Quantification Kit(Catalog No. 15001), which offers greater sensitivity and resistance to many
contaminating agents.

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Protocol II – Fresh or Frozen Tissue
A. Preparation of Nuclear Extract from Tissue
Step 1: Tissue Homogenization
We recommend using only fresh tissue samples. If you have frozen tissue, we suggest you
use the Whole-Cell Extract procedure on page 12. However, nuclear fractionation withfrozen tissue will produce quality nuclear extract, just at a lower yield. Samples should be
kept on ice at all times. All steps are performed on ice or at 4°C with pre-cooled buffers andequipment. It can be generally estimated that there are 2 x 105 cells per milligram of tissue.
However, we highly recommend that you perform an accurate quantitation of cell number foryour specific tissue sample(s) prior to proceeding with the protocol.
Note:

For Complete Lysis Buffer, remember to dilute 1 M DTT stock 1:100 to make 10
mM DTT.

Quick Chart for Preparing Buffers for Nuclear Extraction from Tissue
60mm plate or 100mm plate or
3.2 x 106 cells
8.8 x 106 cells

150mm plateor 2 x 107 cells

Reagents to prepare

Components

1X Hypotonic Buffer (fortissue homogenization)

10X Hypotonic Buffer

400 µL

40 µL

20 µL

Protease Inhibitor Cocktail

40 µL

4 µL

2 µL

Phosphatase Inhibitors

40 µL

4 µL

2 µL

Distilled water

3.5 mL352 µL176 µL1 M DTT4 µL

0.4 µL0.2 µL

Detergent

4 µL

0.4 µL0.2 µL

Total Required

4.0 mL400 µL200 µL

1X Hypotonic Buffer(for cytoplasmic
fractionation)

10X Hypotonic Buffer

25 µL

50.0 µL

100 µL

Distilled water

225 µL450.0 µL

0.9 mL

Total Required

250 µL500 µL

1 mL

Complete Lysis Buffer

10 mM DTT

2.5 µL

5 µL

10.0 µL

Lysis Buffer AM1

22.25 µL

44.5 µL

89 µL

Protease Inhibitor Cocktail

0.25 µL

0.5 µL1.0 µL

Total Required

25 µL

50 µL

100 µL

Total Required

1.25 µL

2.5 µL

5 µL*(Optional) Detergent*The addition of Detergent to the nuclear pellet may help with solubility of proteins, specificallythose tightly associated with membranes or chromatin.

Note:

Volumes of reagents may need to optimized for each tissue.

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1.

Work quickly to preserve tissue during preparation.

2.

For 1 gram of tissue, to obtain a single cell slurry, perform a tissue reduction.
Fresh tissue: Dice tissue into 1-3 mm3 pieces using a clean razor blade or scalpel.
Keep tissue cold by performing this step in a cold ceramic mortar (mortar can be kept
cold by submerging its base in liquid nitrogen). Collect pieces in a pre-chilled, clean
Dounce homogenizer and dounce using a large-clearance (A) pestle.
Frozen tissue: Crush desired amount of frozen tissue using a mortar and pestle
with the base submerged in liquid nitrogen. Some liquid nitrogen can be placedin the mortar bowl along with the frozen tissue. The pestle can be cooled withliquid nitrogen.

3.

This example protocol uses 1 gram of frozen tissue; please adjust the volumes for theamount of tissue used from the chart above. On ice, add 3 mL ice-cold 1X Hypotonic
Buffer containing phosphatase and protease inhibitors supplemented with DTT and
Detergent per gram of tissue and homogenize using the Dounce homogenizer with alarge-clearance (A) pestle for approximately 20 strokes to disrupt tissue. Check under
microscope to ensure that the cells are dissociated, continue homogenizing until you
have a single cell slurry.

4.

Incubate on ice for 15 minutes.

5.

Centrifuge for 10 minutes at 850 x g at 4°C. Transfer the supernatant into a pre-chilled
microcentrifuge tube. The pellet now contains a mixture of unbroken cells and nuclei,
while the supernatant contains part of the cytosolic fraction from any lysed cells.
Optional: The supernatant fraction can be saved to later determine fractionation
efficiency by Western blot if desired.

6.

Most of the cells are still not lysed in the pellet. For cell lysis, continue the procedure
with the pellet at Step 2: Cytoplasmic Fraction Collection, No. 1 on page 8 byresuspending in 500 µL 1X Hypotonic Buffer

B. Preparation of Whole-Cell Extract from Tissue
Quick Chart for Preparing Buffers for Whole-Cell Extraction from Tissue

Reagents to prepare

Components

Complete Lysis Buffer

10 mM DTT

150mm plateor 2 x 107 cells

2.5 µL

5 µL

10.0 µL

Lysis Buffer AM1

22.25 µL

44.5 µL

89 µL

Protease Inhibitor Cocktail

0.25 µL

0.5 µL1.0 µL

25 µL

50 µL

100 µL

Total Required

Note:

60mm plate or 100mm plate or
3.2 x 106 cells
8.8 x 106 cells

Volumes of reagents may need to optimized for each tissue.

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1.

For 1 gram of tissue, perform a tissue reduction by dicing tissue into 1-3 mm3 pieces
using a clean razor blade. Collect pieces in a pre-chilled 15 mL conical tube.

2.

On ice, disrupt and homogenize tissue in 3 mL ice-cold Complete Lysis Buffer
per gram of tissue with a Dounce homogenizer or a Polytron device. Maintaintemperature at 4°C throughout all procedures. Incubate on ice for 30 minutes.

Note:

Frozen tissue can be sliced very thinly and thawed in this buffer. When using
a mechanical homogenizer, begin homogenization at slow speeds until thetissue is broken into smaller pieces and then increase the speed to the
maximum for 45-60 seconds. Avoid the generation of excess heat or foam.

3.

Transfer to pre-chilled microcentrifuge tubes, centrifuge at 10,000 x g for 10 minutes
at 4°C.

4.

Transfer supernatants to new pre-chilled tubes and centrifuge again. Pool
supernatants in the same tube. The supernatant contains the whole-cell lysate.
Sometimes a longer centrifugation is necessary to obtain a clarified lysate.

5.

Aliquot and store at -80°C. Avoid freeze/thaw cycles.
Optional: Save a fraction of the supernatant for determination of extraction efficiency
by Western blot.

Note:

The presence of certain detergents or PMSF may interfere with the Bradford
assay, thus perform a 1:50 or 1:250 dilution of your samples. Use theComplete Lysis Buffer as the blank with a similar dilution as the samples.
Read the absorbance at 595 nm with a plate reader spectrophotometer(refer to the Appendix, Section A on page 14 for an example dilution series).
As an alternative, try Active Motif’s ProStain™ Protein Quantification Kit(Catalog No. 15001), which offers greater sensitivity and resistance to many
contaminating agents.

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Appendix
Section A. Example Dilution for Bradford Protein Quantitation
An example dilution series in preparation for the Bradford Assay is provided below.
1.

To measure the protein concentration utilizing the Bradford Assay, begin with a working
stock of BSA (the example shown below uses working BSA stock at 5 mg/mL).

Note:

2.

The DTT and protease inhibitors in the Complete Lysis Buffer require dilution
before assaying for protein concentration. It is important to add the DTT andprotease inhibitors to the lysis buffer before making the appropriate dilutions.

Dilute the Complete Lysis Buffer in preparation for the assay. The suggested dilutions
are 1:50 or 1:250. Determine number of blank and standard wells you will be assaying
and make enough diluted Lysis Buffer 1:50 or 1:250 stock to perform all dilutions.
Dilute in water. Follow your Bradford Assay instructions for volume needed per well.
Example: Lysis Buffer 1:50 stock = 30 µL Complete Lysis Buffer in 1470 µL H2O
Lysis Buffer 1:250 stock = 6 µL Complete Lysis Buffer in 1494 µL H2O

3.

Make up a 50 µg/mL BSA solution by adding 10 µL of the 5 mg/mL working BSA stock to
990 µL of Complete Lysis Buffer. Next, pipette 0.5 mL of Complete Lysis Buffer into the 4
remaining tubes. Use the 50 µg/mL BSA solution to prepare a dilution series as indicatedbelow. The example standards below use the following concentrations: 50, 25, 12.5,
6.25, and 3.125 µg/mL. Note: The preceding range is provided as guidance, a broader
range of values may be used. Refer to your Bradford Assay instructions for details.
Mix tubes thoroughly before each transfer. Prepare Blank and samples. The 50 µg/mL
standard is the high standard and the Blank (Complete Lysis Buffer alone) is 0.0 µg/mL.
0.5 ml50 µg/ml

0.5 ml25 µg/ml

0.5 ml12.5 µg/ml

0.5 ml6.25 µg/ml

3.125 µg/ml

Example: 10 µL BSA (5 mg/mL) in 0.99 mL Complete Lysis Buffer 1:50 or 1:250 stock = 50 µg/mL
		Transfer 0.5 mL into 0.5 mL Complete Lysis Buffer 1:50 or 1:250 stock = 25 µg/mL
		Transfer 0.5 mL into 0.5 mL Complete Lysis Buffer 1:50 or 1:250 stock = 12.5 µg/mL
		Transfer 0.5 mL into 0.5 mL Complete Lysis Buffer 1:50 or 1:250 stock = 6.25 µg/mL
		Transfer 0.5 mL into 0.5 mL Complete Lysis Buffer 1:50 or 1:250 stock = 3.125 µg/m
		
		
		

4.

Blank = Complete Lysis Buffer 1:50 or 1:250 stockStandards = dilute BSA into Complete Lysis Buffer 1:50 or 1:250 stock
Samples = 1 µL sample diluted into 49 µL or 249 µL water

Analyze Blank, Standards and Samples by measuring the absorbance at 595 nm witha spectrophotometer according to your Bradford Assay instructions.
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14Section B. Troubleshooting Guide
Problem / Question

Possible cause

Recommendation

Low protein
concentration incytoplasmic fraction

Volumes of extraction
reagents not appropriate
for given number of cells

Adjust volumes of reagents as indicated in the
Quick Chart for Preparing Buffers.

Plasma cell membrane
not disrupted in cell pellet
in Step 2, No. 1 of the
Preparation of Nuclear
Extract from Cells ofProtocol I (page 7)

Gently pipette up and down to disrupt cell pellet.
Monitor lysis under microscope. Use Dounce
homogenizer with small-clearance (B) pestle.

Incorrect volume of
Lysis Buffer or starting
cell number

Decrease volume of Lysis Buffer or increase
number of cells.

Volumes of extraction
reagents not appropriate
for given number of cells

Adjust volumes of reagents as indicated in the
Quick Chart for Preparing Buffers.

Nuclear proteins lost incytoplasmic fraction after
rupture of nuclei

Monitor under microscope. Reduce vortex,
centrifuge force and time in Step 2, Nos. 2 and 3of the Preparation of Nuclear Extract from Cells
section of Protocol I (page 7). Stop incubation
when nuclei are released.

Nuclear pellet notdispersed in Step 3, No.
1 of the Preparation of
Nuclear Extract from Cells
of Protocol I (page 7)

Vortex thoroughly to ensure nuclear lysis. Use
the Dounce homogenizer if the pellet hastrouble solubilizing.

Cell type is not compatible
with extraction procedure

Swelling and lysis conditions and reagents need
to be optimized for this cell type.

Overlysis

Nuclear proteins are present in cytoplasmic
fraction. Reduce incubation time in Hypotonic
Buffer. For frozen samples, cytoplasmic
fraction will have proteins due to nuclear lysis
during freeze/thaw.

Incomplete lysis

Cytoplasmic proteins are present in the nuclear
fraction. Check cells under a microscope after
adding Detergent for presence of free nuclei
indicative of efficient lysis. Alternatively, perform
10 strokes with a chilled Dounce homogenizer
with a small-clearance (B) pestle to completely
lyse cells.

Low protein
concentration in
nuclear fraction

No or low protein
yield in either
cytoplasmic or
nuclear fractions

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Problem / Question

Possible cause

Recommendation

Poor protein
compartmentalization

Incomplete removal ofcytoplasmic fraction

Make sure to remove all cytoplasmic fraction fromthe nuclear pellet before adding Lysis Buffer.

No or low
protein activity

Collected proteinsare degraded

Maintain low temperature requirements during
procedure. Limit procedure time to a minimum
and snap freeze aliquots right away. Add more ordifferent protease inhibitors.

Nuclear pellet isviscous and will
not resuspend

Incomplete dispersal of
nuclear pellet

Vortex thoroughly to ensure nuclear lysis. Use
the Dounce homogenizer if the pellet has trouble
solubilizing. Certain cell types produce a viscous
nuclear pellet and still produce efficient yield fromthe nuclear extraction.

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16

Technical Services

China

Japan

EuropeNorth America

If you need assistance at any time, please call or send an e-mail to Active Motif Technical
Service at one of the locations listed below.

Toll free:877 222 9543

Direct:

760 431 1263

Fax:

760 431 1351

Email:

tech_service@activemotif.com

Direct:+32 (0)2 653 0001

Fax:

+32 (0)2 653 0050

Email:

eurotech@activemotif.com

Direct:+81 (0)3 5225 3638

Fax:

+81 (0)3 5261 8733

Email:

japantech@activemotif.com

Direct:(86)-21-20926090

Cell Phone:
Email:

18521362870
techchina@activemotif.com

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