AMR 141 ABRF poster 12/15/99 11:48 AM Page 1

2 -D Electrophoresis
sample Preparation
A high-quality 2-D gel has well-defined spots and little streaking in Cell Disruption Methods
Method Application
either direction. Achieving that quality depends on the quality of the Osmotic lysis Blood and tissue culture cells: useful when further subcellular fractionation is desired.

sample applied to the first dimension gel. Freeze-thaw lysis Bacterial and tissue culture cells
Detergent lysis Tissue culture cells
The ideal sample preparation protocol will render all proteins quantitatively soluble, disaggregated, denatured and
reduced in a non-ionic solution. Real protocols, however, must strike a balance between qualitative and quantita- Enzymatic lysis Plant tissue, bacteria and fungi: use pectinase, cellulasae, lysozyme or lyticase™, e.g., to remove cell walls.

tive performance. When we select a sample preparation protocol, we must consider the specific goals of the pro- Sonication Cell suspensions
ject: Which is more important—to see as many proteins as possible, streaks and all; or to generate a clear and French Press Cell Microorganisms with cell walls
reproducible, but not necessarily complete, pattern of the proteins associated with a particular sample? Each step Grinding Solid tissue, microorganisms: mortar and pestle
taken to improve the quality of the gel risks selective loss of proteins from the sample. Thus there is always a Mechanical homogenization Solid tissue: Dounce or Potter-Elvehjem homogenizers, blenders
tradeoff between gel quality and sample completeness.
Glass bead homogenization Cell suspensions, microorganisms: vortex in presence of glass beads

In preparing a sample for 2-D analysis a number of factors must be considered: Protease Inhibitors
Inhibitor Effective Against Limitations
• Does the cell disruption method release the proteins of interest?
• Does the disruption damage or degrade proteins in the sample? PMSF Serine- and some cysteine proteases Very toxic. Rapidly inactivated in aqueous solutions by DTT
(Phenylmethylsulphonyl fluoride) or 2-mercaptoethanol
• Is some form of protease inhibitor needed to prevent proteolysis?
AEBSF (Pefablock) ™ Serine- and some cysteine proteases May covalently modify protein, affect pl. More soluble, less
• Are interfering substances present that must be removed? (Aminoethylbenzylsulphonyl fluoride) toxic than PMSF
• Are the proteins completely soluble under electrophoretic conditions? EDTA or EGTA Metalloproteases
• Does the composition of the sample solution significantly affect the pI of the protein or leave the sample in a
TLCK, TPCK Many serine- and cysteine proteases
highly conductive solution? (Tosyl lysine chloromethyl ketone, tosyl
phenylalanine chloromethyl ketone)
Due to the diversity of protein samples, the best combination of steps and reagents usually must be determined
Benzamidine Serine proteases
empirically for each sample type. Commonly, 2-D experiments begin with extraction into a solution containing at
least 7 M urea and at least 2% CHAPS. Other treatments and additives are then evaluated if additional optimiza- Peptide Protease Inhibitors Serine-, cystine-, aspartyl-proteases, Expensive, small peptides that may show up on gels
(leupeptin, pepstatin, aprotinin bestatin) aminopeptidases
tion is necessary. Here we address some of the more common 2-D sample preparation issues. Additional details
and suggestions are covered in our handbook 2-D Electrophoresis Using Immobilized pH Gradients: Principles
and Methods, which is available from Amersham Amersham Biosciences (80-6429-60).

Contaminants That Affect 2-D Results Effect of TCA/acetone precipitation Effect of salt on 2-D separations
Lyophilized E. coli cells were extracted into 8 M urea, 4% CHAPS by sonication. The lysate was Lyophilized E. coli cells were extracted into 8 M urea, 4% CHAPS by sonication. The lysate was
Contaminant Material Removal Techniques cleared by centrifugation and a portion of the lysate was precipitated with 10% trichloroacetic cleared by centrifugation and focused in the absence (left) or presence (right) of 30 mM NaCl. The
acid (TCA), 80 % acetone then resuspended in 8 M urea, 2% CHAPS. The samples were focused first dimension was run on 13 cm long Immobiline DryStrip pH 4-7 gels.
Salts, buffers, endogenous Dialysis, spin dialysis, gel filtration,or precipitation on 13 cm long Immobiline™ DryStrip pH 3-10 NL gels.
small ionic molecules and resuspension
Left: precipitated and redissolved extract. Right: untreated extract
Ionic detergents (SDS) Dilute the SDS-containing sample in a rehydration solution
containing a zwitterionic or non-ionic detergent so final
concentration of SDS is ≤ 0.25% and the ratio of detergent
to SDS is ≥ 8:1.

Nucleic acids (DNA, RNA) Treat sample with DNase/RNase mixture. Note, DNase and
RNase may appear on 2-D map.

Polysaccharides Precipitate sample in TCA, ammonium sulphate or phenol/

ammonium acetate, then centrifuge. Ultracentrifugation may
remove high MW polysaccharides.

Lipids Precipitation with acetone removes some lipids. Strongly

denaturing conditions and detergents minimize protein-lipid
interactions. Excess detergent may be needed.
Phenolic compounds Use reducing agents during extraction. Rapidly separate
proteins from phenolic compounds by precipitation tech-
niques.
Insoluble material Always clarify samples by centrifugation prior to application
to first dimension.
TCA/acetone precipitated Control
Conclusion: Precipitation with TCA/acetone decreases the streaking associated with endogenous
small ionic molecules. However, difficulty in solubilizing the pellet can decrease total protein
recovery and result in loss of some proteins.
SDS extraction
Extracts of third instar larvae of Drosophila melanogaster were prepared by homogenization
either into solution containing 8 M urea, 4% CHAPS (left) or into 2% SDS followed by heating
at 100 °C for 3 min (right). Both samples were subsequently diluted into rehydration solution
containing 8 M urea, 2% CHAPS, 0.5% IPG Buffer, 60 mM DTT. The samples were focused on
13 cm long Immobiline DryStrip pH 3-10 NL gels.
0 mM NaCl 30 mM NaCl
Conclusion: First dimension focusing in the presence of salt was slower, as the maximum attain-
able voltage was considerably lower. The presence of salt resulted in streaking, gaps in the pat-
tern and absence of focusing in parts of the pH range.
Effect of DNA on 2-D separation
Lyophilized E. coli cells were extracted into 8 M urea, 4% CHAPS by sonication. Each sample
contained 32 µg of protein and 2.7 µg of added DNA. One of the samples was treated with
DNase I. The first dimension was run on 7 cm long Immobiline DryStrip pH 3-10 NL gels
Left: DNase treated extract. Right: untreated extract

Protein Solublization Agents

Component Functions
Denaturants
Urea (8–9.8 M) Concentrations up to 9.8 M can be used.

Urea (7 M) + Thiourea (2 M)
A mixture of urea and thiourea may be superior to urea
alone for solubilizing some proteins (especially membrane
proteins). Thiourea may require deionization or recrystalization
to remove interfering contaminants. Note: thiourea is a
suspected carcinogen.
8 M Urea, 4% CHAPS extraction Hot 2% SDS extraction
Conclusion: Extraction with hot 2% SDS can in some instances result in a significantly better
DNase treated Untreated
2-D separation than extraction into a urea/CHAPS mixture. This method is only possible when
Conclusion: Excessive DNA contamination resulted in decreased resolution and increased back-
the protein concentration in the extract is sufficient to allow dilution into a significant excess ground staining at the acidic end of the separation.
of CHAPS-containing rehydration solution.

Detergents
CHAPS (2–4 %) CHAPS is the zwitterionic detergent used most commonly.

Triton™ X- 100 or NP 40 Non-ionic detergents Effect of denaturants Effect of reductant

Extracts of rat liver were prepared either by homogenization into solution containing 4% CHAPS, Lyophilized beef liver was extracted with 8 M urea, 4% CHAPS and with either 60 mM DTT
Sulfobetaines Sulphobetaines are zwitterionic detergents that are
2 mM TBP, 40 mM Tris and 1 mM PMSF with either 7 M urea and 2 M thiourea (left) or 8 M (dithiothreitol, left), 4 mM TBP (tributyl phosphine, middle) or no reductant (right). Each sample
(e.g. Zwittergent™, SB 3-10) especially effective at solubilizing membrane proteins. They
urea (right). The samples were focused on 13 cm long Immobiline DryStrip pH 3-10 NL gels in a was precipitated with TCA and acetone and resuspended in 8 M urea, 4% CHAPS, 0.5% Pharm-
are not compatible with urea in concentrations over 5M,
rehydration solution containing 2% CHAPS, 60 mM DTT and 0.5% Pharmalyte 3-10 with either alyte 3-10 and the same reductant as in the extraction. The first dimension was run on 13 cm
which limits their utility in sample preparation for 2-D.
7 M urea and 2 M thiourea (left) or 8 M urea (right). long Immobiline DryStrip pH 3-10 NL gels in a rehydration solution containing 8 M urea, 2%
Amidosulphobetaines These strongly solubilizing detergents can be used together CHAPS, 60 mM DTT and 0.5% Pharmalyte 3-10.
(e.g. ASB 12, ASB 14) with 8 M urea or 7 M urea, 2 M thiourea.

SDS (sodium dodecyl sulphate) SDS can be useful in extracting and solubilizing proteins for
2-D electrophoresis, but the sample must subsequently be
diluted into a solution containing at least an 8-fold excess
of non-ionic or zwitterionic detergent prior to IEF.

Reductants
DTT (dithiothreitol) Cleaves disulphide bonds, allowing proteins to unfold
completely, and prevents oxidative aggregation.
60 mM DTT 4 mM TBP No reductant
2-ME (2-mercaptoethanol) Commonly used reductant, very noxious. Requires high Conclusion: The choice of reductant used in preparing the sample (or whether one was used
concentrations that may cause artifacts from inherent at all) had little effect on the 2-D result.
impurities. (Also referred to as β-mercaptoethanol.) 7 M urea + 2 M thiourea 8 M urea

Conclusion: Thiourea improves extraction efficiency, particularly of high molecular weight pro-
TBP (tributyl phosphine) Tributyl phosphine is an uncharged reductant that may be
teins. However, there is increased streaking between pH 4-5 in the presence of thiourea.
more effective than DTT in some situations. Note: TBP is
quite toxic, gives off noxious fumes and can ignite if it Effect of buffer or carrier ampholytes
comes in contact with dry organic materials. Extracts of rat liver were prepared either by homogenization into solution containing 8 M urea, 4%
Effect of detergent CHAPS and 60 mM DTT with either 0.5% Pharmalyte 3-10 (left) or 20 mM diethanolamine-HCl pH
9.0 (right). The samples were focused on 13 cm long Immobiline DryStrip pH 3-10 NL gels.
Spinach thylakoid membranes were solubilized in a solution containing 7 M urea, 2 M thiourea,
0.5% Pharmalyte 3-10 and 60 mM DTT plus either 2% CHAPS (left) or 2% amidosulphobetaine
C12 (right). The samples were focused on 13 cm long Immobiline DryStrip pH 3-10 NL gels in a
Amersham BiosciencesUK Limited rehydration solution containing 7 M urea, 2 M thiourea, 60 mM DTT and 0.5% Pharmalyte 3-10
Amersham Place Little Chalfont Buckinghamshire England HP7 9NA
with either 2% CHAPS (left) or 2% amidosulphobetaine C-12 (right).
Amersham Biosciences AB
SE-751 84 Uppsala Sweden
Amersham Biosciences Inc.
800 Centennial Avenue PO Box 1327 Piscataway NJ 08855 USA
Amersham Biosciences Europe GmbH
Munzinger Strasse 9 D-79111 Freiburg Germany

www.amershambiosciences.com

Immobiline and Pharmalyte are trademarks of Amersham Biosciences

imitedand its subsidiaries. Pefablock is a trademark of
Boehringer-Mannheim Corporation. Triton and Tris are trademarks of
Union Carbide Chemicals and Plastics Company. Zwittergent is a
trademark of Calbiochem-Novabiochem Corporation. Amersham
Amersham Biosciences is a trademark of Amersham plc.

© 1999 Amersham Amersham Biosciences Inc. All rights reserved.

All goods and services are sold subject to the terms and conditions 0.5% Pharmalyte 3-10 20 mM diethanolamine-HCl pH 9.0
of sale of the company within the Amersham Amersham Biosciences
group that supplies them. A copy of these terms and conditions Conclusion: Extraction in the presence of carrier ampholyte and extraction in the presence of high
is available on request.
Printed in the USA.
2% CHAPS 2% Amidosulphobetaine C-12 pH buffer are both commonly performed. In this experiment, both treatments resulted in high quality
80-6448-60/Rev. AO/ 12-99 2-D patterns with many spots. However, the spot profiles were significantly different. Some proteins
Conclusion: Either zwitterionic detergent results in essentially the same 2-D pattern. A few were preferentially solubilized in the presence of carrier ampholyte and others were preferentially
additional spots are visible when amidosulphobetaine C-12 is used in the solubilization (circled solubilized in the presence of pH 9.0 buffer.
in red). The use of amidosulphobetaine C-12 also results in increased vertical streaking and
silver staining background.