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800-462-3417
Suggestions for Sample Preparation for 2DE
Index
I. Introduction and General Instructions........ 1-2
A. How much protein should be loaded
B. Total amount required
C. What sample buffer should be used
II. Sample Buffers ......................................... 2-3
A. Urea Sample Buffer
B. SDS Boiling Buffer
C. SDS Boiling Buffer minus BME
D. Osmotic Lysis Buffer
E. 100× Protease Inhibitor Stock Solution
F. 10× Nuclease Stock Solution
G. OmniCleave™ Endonuclease
H. Phosphatase Inhibitors
III. 2D Pattern Visualization .......................... 3-4
A. Coomassie blue versus silver stain
B. Sypro ruby, other fluorescent stains
C. Fluorography
D. Autoradiography
IV. Preparing Samples.................................. 4-5
A. In Urea Sample Buffer
B. In SDS Boiling Buffer
C. Cultured cells
D. Yeast, S. aureus and difficult-to-lyse cells
E. Immunoprecipitates
F. Animal tissue
V. Determining Protein Concentration ..............5
VI. Protein Precipitation Methods…………….5-6
A. Ethanol Precipitation
B. TCA in acetone
C. Phenol/NH4Acetate for plant samples
D. Chloroform/methanol to remove lipids
VII. Determining Protein Bound Dpm ........... 6-7
VIII. Radiolabeling Cells .............................7
A. 35S-labeling of cultured cells
B. 32P labeling of cultured cells
IX. Mailing Samples..........................................8
X. Reference List ..............................................8
1
For advice: call 800-462-3417 (locally 608258-1565) or email [email protected]
I. Introduction
The quality of 2D gels returned to you is highly
dependent upon sample preparation. Generally
sample preparation is as simple as sending us
samples in SDS or urea buffer dissolved to a known
protein concentration. Problems that arise because
of high salt, protein insolubility or dilute concentration can usually be remedied. Following these
suggestions will help you stay away from unfixable
problems such as protease degradation, and
produce the best possible results.
Changing sample preparation may alter 2D patterns
so once you decide on a protocol, stick with it. If you
are uncertain, try your protocol on one or two
samples before proceeding to larger numbers.
If you have questions please email or call. We know
your samples are precious and will do our very best
to make your project successful.
This guide is online at www.kendricklabs.com, see
2D Services (dropdown menu) Sample Preparation
General Instructions
Start by filling out the sample identification form from
our web site (2D services dropdown menu, 5th
item). You’ll have to decide how much sample to
load, the gel staining method and method of
analysis. Call or email for advice, or you can leave
undecided lines blank and let our Lab Manager
figure it out (he’ll call you with any questions.)
A. How much protein should be loaded?
1.Complex samples like whole cell lysates and
cytosols are usually run on large format (LF, 20 x
22 cm) 2D gels. For LF, a 100 ug load is
recommended for silver and a 600 μg load for
Coomassie blue staining. The maximum volume that
can be loaded is 150 ul. Cytosolic protein mixtures
give cleaner patterns than whole cell lysates. For
standard format (SF, 13 x 15 cm) 2D gels load 50
ug for silver and 200 ug for Coomassie staining.
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2. Purified proteins are run on SF 2D gels. For
Coomassie blue we suggest a load of about 2 ug/
spot and for silver staining about 100 ng/spot. If in
doubt triple the load. Overloading does not severely
affect IEF of semi-purified proteins. Glycoproteins
tend to focus over a broad pH range and should be
loaded at 10−20 μg protein for Coomassie and 1−2
ug for silver.
3. Membrane fractions may be run on either LF or
SF gels depending on the sample. Lipids, polysaccharides, and polynucleotides in membrane samples
cause streaks and decrease resolution and are the
limiting factor. Nucleic acid fragments stain strongly
with silver and may cause high background. Call for
consultation about which gel size to use. In either
case it is best to use silver stain and load 75 ug for
SF gels, 150 ug for LF. If Coomassie blue staining is
necessary for mass spectrometry we suggest 100 ug
maximum load for SF and 400 ug for LF. When in
doubt it is best to titrate the load up rather than going
for broke when trying to load maximally.
4. Other subcellular fractions, nuclear matrix for
example [1], can be isolated as described in the
literature and run on SF 2D gels. If enough material
is available we will optimize gel conditions at no
extra charge for such samples.
5. Samples for Transblotting: For Western blotting
it is best to load the maximum amount possible on
SF 2D gels. For complex samples such as whole cell
lysates, the optimal load is 200 ug for both transblotting and duplicate 2D gels for mass spectrometry.
Since 50 ul is the maximum loading volume, the
sample must be >4 mg/ml protein.
B. Total amount required
If possible we would like to receive four times the
recommended load to allow for free repeats in case
initial problems arise. For purified proteins, please
include enough for two to four runs (call or email to
discuss). If your samples are too dilute, you may
concentrate by lyophilization (but be careful of
concentrating salts) or by protein precipitation.
Desired counts per minute (cpm)/μl for complex
radiolabeled samples are: > 25,000 for 35S and 14C
for direct autoradiography, > 8000 for 35S or 14C for
fluorography, and > 4,000 for 32P direct
autoradiography. The counts must be protein bound.
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C. What sample buffer should be used?
We use two buffers to dissolve samples: Sodium
Dodecyl Sulfate (SDS) Boiling Buffer and Urea
Sample Buffer. Recipes are given on the next page.
About 95% of samples run at Kendrick Labs are
dissolved in SDS buffer or in SDS:Urea buffer 1:1.
A huge advantage to using carrier ampholines with
IEF tube gels instead of IPG strips is that SDS can
be used in sample preparation [2]. Although SDS is
negatively charged it is stripped from the proteins
during isoelectric focusing (IEF) to form micelles with
the nonionic detergent IGEPAL [3], formally known
as Nonidet P-40. The micelles migrate to the acidic
end of the tube gel and form a bulb.
Note: if your proteins of interest are basic, with
pIs > 9.0, they will require non-equilibrium pH
gradient 2D electrophoresis (NEPHGE), which is
incompatible with SDS [4]. Samples for NEPHGE
must be dissolved in Urea Sample Buffer.
Advantages of SDS: Heating biological samples in
the presence of SDS dissolves proteins more
completely than any other method. This is especially
true for membrane proteins. SDS sharpens 2D spots
and increases the recoveries of most proteins in the
gel. 2D gel patterns obtained in the presence of SDS
are similar but not identical to those obtained with
Urea Sample Buffer without SDS. However, they are
quite reproducible. Sample preparation with SDS
Buffer is much easier than with Urea Buffer. There
are no undissolved pellets to discard.
Disadvantages of SDS: The acidic end of the tube
gel (as much as 1.5 cm) becomes distorted due to
formation of the SDS/IGEPAL bulb. To correct for
this we run longer IEF tube gels for SDS-containing
samples and remove the bulb, which contains no
resolvable proteins. The remaining tube gel is the
normal length.
Isoelectric point measurements may be unreliable in
the presence of SDS because residual detergent
may remain on some of the proteins. A few proteins
become streaky in the presence of SDS.
II. Sample Buffers
The following gives formulas and references for
various sample buffers. SDS and urea from different
suppliers may give different 2D patterns so don’t
switch reagents in the middle of a project.
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We recommend SDS from Shelton Scientific
(Shelton, CT) cat # IB07062 and ultrapure urea from
MP Biomedicals (Aurora, OH) cat # 821519.
A. Urea Sample Buffer [3] contains 9.5 M urea,
2% w/v IGEPAL CA-630 (a non-ionic detergent, or
Nonidet P-40), 5% beta-mercaptoethanol (BME),
and 4% Pharmalytes pH 3-10 (GE Healthcare,
Piscataway, NJ). Ten 1 ml aliquots of this buffer are
supplied in the mailing kit.
B. SDS Boiling Buffer [5] contains 5% SDS, 5%
BME, 10% glycerol and 60 mM Tris, pH 6.8. Protein
in solution at a final concentration of 35 mg/ml or
less may be heated to boiling in this buffer to aid
dissolution. Ten 1 ml aliquots of this buffer are
supplied in the mailing kit.
C. SDS Boiling Buffer Minus BME works best
to dissolve protein pellets from cultured cells and
other hard-to-dissolve samples prior to protein
determinations using the BCA assay [6]. Note: SDS
inhibits nuclease activity so polynucleotide digestion
must be carried out prior to its addition. Ten 1 ml
aliquots are supplied in the mailing kit.
D. Osmotic Lysis Buffer [7] contains 10 mM Tris,
pH 7.4, and 0.3% SDS. Proteins in solution at a final
concentration of 2.0 mg/ml or less may be heated to
boiling in this buffer. Note: there are no sulfhydryl
reducing agents (BME, DTT) in OLB so the BCA
protein assay may be performed. Ten 1 ml aliquots
are supplied in the mailing kit.
E. 10× Nuclease Stock Solution [8] contains 50
mM MgCl2, 100 mM Tris, pH 7.0, 500 μg/ml RNase
(Ribonuclease A from bovine pancreas Type IIIA,
Sigma R5125) and 1000 μg/ml DNase
(Deoxyribonuclease I, Type II from bovine pancreas,
Sigma D4527). Final concentrations for these
enzymes should be 50 μg/ml RNase and 100 μg/ml
DNase in 5 mM MgCl2 and 10 mM Tris-Cl, pH 7.0.
Ten 100 μl aliquots are supplied in the mailing kit.
F. 100× Protease Inhibitor (PI) Stock Solution
[9-11] contains 20 mM AEBSF (Calbio-chem
101500), 1 mg/ml leupeptin (Sigma L2884), 0.36 mg/
ml E-64 (Sigma E3132), 500 mM EDTA (Calbiochem
34103), and 5.6 mg/ml benzamidine (Sigma B6506).
This stock solution should be added to samples at a
final concentration of 1%. Two 100 μl aliquots are
supplied in the mailing kit.
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G. OmniCleave™ Endonuclease from Epicentre
Technologies (Madison, WI, # OC7810K) is a
purified enzyme from a recombinant E. coli strain
that works in the presence of SDS. It reduces
sample viscosity by degrading native DNA and RNA
to di-, tri- and tetranucleotides. Dilute to 20 unit/ul
with the dilution buffer supplied, i.e. add 0.5 ml to the
tube of 10 kU, and use as needed.
H. Phosphatase Inhibitors should be added for
experiments involving protein phosphorylation.
Phosphatase Inhibitor cocktails may be purchased
from EMD Biosciences (Novagen) cat. # 524624
(inhibits protein serine/theonine phosphatases) and
#524625 (inhibits protein tyrosine phosphatases).
III. 2D Pattern Visualization
A. Coomassie blue versus silver stain
Coomassie blue staining can detect as little as 0.05
μg/polypeptide spot. It is a quantitative stain [5] and
is fully compatible with mass spectrometry. Silver
staining is much more sensitive and can detect 5 ng/
poly-peptide spot but is semiquantitative because
different proteins saturate at different levels [12]. A
“special” silver stain is also offered for mass
spectrometry of bacterial samples with a detection
limit of ~10 ng/spot [13]. Note that proteins often
stain negatively with special silver so quantification is
not possible.
B. Sypro Ruby, Other fluorescent stains
We are glad to stain your gels with fluorescent stains
such as Sypro ruby, Cy dyes for DIGE, Pro-Q
Diamond, etc., digitize the images with a Bio-Rad FX
Pro Plus Multi-Imager and analyze the patterns with
Progenesis software. Or the stained gels can be
returned to you either wet or dry.
C. Fluorography
Fluorography [14] is a method in which radioactive
slab gels are permeated with a fluor prior to
exposure to x-ray film at -70°C. This reduces film
exposure time for 35S and 14C to one fifth of that
required for direct autoradiography. We recommend
“ENHANCE” from New England Nuclear as the
fluorographic agent.
Fluorography is often used for 35S- and 14C-labeled
samples and almost always for 3H. The major
disadvantage is that the fluor causes a faint mottling
throughout the 2D pattern.
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Coomassie blue or silver staining prior to
fluorography causes some quenching but enables
the pattern to be matched to a duplicate Coomassie
2D gel for mass spectrometry.
We suggest a load of at least 500 dpm for purified or
semi-purified proteins. Quite often proteins appear
as a set of charge isomers with the counts spread
over a wide area. When in doubt load several
thousand counts to visualize minor components. For
protein mixtures such as cultured cells labeled with
14
C or 35S, load 350,000 dpm for 2–4 day film
exposures. Specific activities greater than 30,000
dpm/μg are recommended. Load 1 uCi 3H for a 2 day
fluorographic exposure.
D. Autoradiography
Whenever possible, direct autoradiography is
preferable to fluorography, especially for quantification. For purified proteins and immunoprecipitates,
loads of 2500 dpm for 32P and 5000 dpm for 14C or
35
S are recommended. For complex patterns, loads
of 0.8-1.0 uCi 35S or 14C, and 200,000 dpm 32P are
recommended for a 2 day film exposure at room
temperature. 3H-proteins can be transblotted to
PVDF before exposure.
IV. Preparing Samples
High concentrations (> 150 mM) of NaCl, KCl and
other salts cause serious streaking problems as do
lower concentrations of phosphate and buffers. Keep
salt concentrations as low as possible. Dialyzing
samples to remove salts and other low molecular
weight substances is recommended. Lyophilization
of dilute samples prior to adding sample buffer is
ideal, do not concentrate salts greater than 150
mM. Beware of high viscosity due to concentration of
non-ionic substances such as sucrose.
If there is a precipitant in the dissolved sample, pellet
it by centrifugation. Precipitants tend to impede
isoelectric focusing by plugging the top of the IEF
tube gel.
A. In Urea Sample Buffer
Urea (H2NCONH2) is commonly used as a
denaturant to solubilize proteins for isoelectric
focusing, especially for purified and semi-purified
proteins. Although urea does not dissolve proteins
as well as SDS, there is no danger of it changing
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800-462-3417
the apparent isoelectric point. It is often convenient
to dilute a concentrated protein solution 1:1 or 2:1
with Urea Sample Buffer or simply to dissolve the
lyophilized protein powder in the buffer. If the
sample does not dissolve well try heating it to 50oC
for 10 min or freeze/thawing several times. NEVER
BOIL SAMPLES IN UREA, isocyanates from the
urea will react with the proteins to form charge
isomer artifacts [15].
B. In SDS Boiling Buffer
Add SDS Boiling Buffer to the sample, vortex
thoroughly, and place the tube in a boiling water bath
for 5 minutes [16]. For example, if you have 320 ug
protein in a microcentrifuge tube after lyophilization
and want to load 150 ug in 30 μl, add 64 μl SDS
Boiling Buffer, tap lightly to dissolve the powder,
place in a boiling water bath for 2-5 min, and freeze.
Alternatively, dilute the samples 1:1 or 2:1 with SDS
Boiling Buffer prior to heating. The 5% SDS in the
buffer is sufficient to solubilize a protein concentration of 35 mg/ml based on the rule of 1.4 g SDS per
gram of protein [17].
C. Cultured cells
Remember to work quickly on ice to avoid
proteolysis. Have Osmotic Lysis Buffer, 10×
Nuclease Stock solution, 100x Protease Inhibitor (PI)
Stock solution, phosphatase inhibitor cocktails (when
necessary), SDS Boiling Buffer + BME (or Urea
Sample Buffer), prelabeled microcentrifuge tubes,
and dry ice ready. Prepare Osmotic Lysis Buffer by
adding 100 ul of Nuclease Stock solution and 10 ul
of PI stock/ml plus phosphatase inhibitor cocktails if
required. Rinse the cells 3 times with cold buffered
saline. Aspirate the excess liquid.
For plated cells, add prepared Osmotic Lysis Buffer
directly to the cells on the cold dish. Try to add a
volume to give 2–4 ug protein/μl. A 35 mm diameter
dish containing 40,000 cells/cm2 (approximately
380,000 cells) would receive 50 ul of Osmotic Lysis
Buffer. If the dish contains 400,000 cells/cm2, then
add 150 ul of Osmotic Lysis Buffer. While keeping
the dish on ice, scrape the cells from the dish using
a rubber policeman and mix them with the buffer.
Transfer to a 1.5 ml microcentrifuge tube, vortex and
incubate on ice for 5–30 min. The viscosity due to
DNA and RNA should quickly disappear. If it doesn’t,
add 2 ul of OmniCleave™ or sonicate the cells for 5
min. Repeatedly draw the samples through a smallbore needle until the samples can be pipetted.
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For suspended cells, pellet the cells by centrifugation
and rinse them twice in cold, buffered saline by
resuspending and centrifuging to remove serum
proteins and/or unbound radiolabel. The final rinse
should be in a 1.5 ml microcentrifuge tube. Estimate
the amount of Osmotic Lysis Buffer to be added to
give 2–4 ug protein/μl. Use 40–50 ul per 2–4 million
cells. Add prepared Osmotic Lysis Buffer to the
pellet and vortex thoroughly. Use a pipette to
resuspend if necessary. Vortex and incubate on ice
for 5–30 min. If the mixture remains viscous, add 2
ug OmniCleave and or sonicate the cells for 5 min.
Remember to reserve an aliquot at this stage if you
wish to determine protein concentration using the
BCA method, or to determine protein-bound counts
as described in Section VII. The 0.3% SDS in the
Osmotic Lysis Buffer, which helps to solubilize
proteins and inhibit proteases, will not interfere with
the BCA method. If the cells or pellets aren’t
dissolving, add more SDS Boiling Buffer minus BME
and place the tubes in a boiling water bath for 5 min.
You may still do protein determinations on < 20 uL
aliquots using the BCA method.
At this point, add an equal amount of SDS Boiling
Buffer containing BME or just add BME to 5%
depending on previous steps and boil for 5 min.
Alternatively, add an equal amount of Urea Sample
Buffer and heat to 50°C for 1 min. Do not boil
samples in Urea Sample Buffer; do not add SDS
Buffer to samples requiring NEPHGE. Store the
samples at -70°C until mailing or pickup.
D. Yeast, S. aureus & difficult-to-lyse cells
To each sample of gently pelleted cells, add 500 ul
of osmotic lysis buffer containing PI Stock, nuclease
stock, phosphatase inhibitor stock if needed, and
100 ug of washed glass beads (Sigma G9268, mesh
size 425–6000 microns) per 50–100 ul washed cell
pellet as described by Jazwinski [18]. Vortex sample
thoroughly, freeze, centrifuge, and repeat until the
pellet size has been substantially reduced. Add 400
μl of SDS Boiling Buffer minus BME, vortex, and
freeze again. Place the tube in a boiling water bath
for 5 min then centrifuge. Lyophilize the supernatant;
remember to reserve an aliquot for protein
determination. Dissolve the resulting residue in 1:1
diluted SDS Boiling Buffer to at least 5.0 mg/ml for
Coomassie blue-stained gels or 1.0 mg/ml for silverstained gels.
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E. Immunoprecipitates e.g. Brown et. al. [19] The
amount of protein is small for IPs and there is no
danger of overloading the gel. Generally the amount
of protein stripped off of affinity beads for 2D should
be 3-4 times that required for a 1D gel, since bands
often are resolved into a string of spot isoforms.
Duplicate Coomassie blue stained gels may be run
in tandem and matched to the Western blot film for
mass spectrometry. Generally as much sample as
possible is loaded on the Coomassie blue gel. To
ship IP’s on beads, remove the supernatant and
send the bead pellet on dry ice by express mail. Our
Lab Manager will consult with you and then do
further sample preparation at no charge.
F. Animal Tissue [20]
Weigh the tissue, freeze to -80°C, crush with mortar
and pestle then place in a tissue homogenizer on
ice. Add about 0.25 ml of Osmotic Lysis Buffer
containing PI Stock, Nuclease Stock and
Phosphatase inhibitor stock/100 mg tissue and
homogenize on ice. Freeze/thaw twice. Allow the
nucleases to react for 10–15 min on ice, add an
equal amount of SDS Boiling Buffer - BME and place
in a boiling water bath for 5–30 min. Cool the tissue
on ice, centrifuge to pellet solids, determine protein
concentration, and store at -70°C.
V. Determining Protein Concentration
There are four common spectrophotometric methods
of determining protein concentration: BCA method
[6] Lowry method [21], Bradford method [22], and
absorbance at A280/A260 [17]. Reagents and protocols
for the first three can be obtained respectively from
Pierce Chemicals, Sigma Chemical Co., and BioRad Labs. We will accept protein determinations
from any of these methods but would like to know
which method was used so we can adjust the loads.
The Bradford method tends to give higher values for
mixtures than the values obtained from the BCA and
Lowry methods.
We recommend the BCA method because it has
good standard curve stability and relatively few
compounds interfere (Pierce provides a list including
1% SDS and 3.0 M urea). Note, however, BME and
DTT strongly interfere with the BCA assay.
Remember to take aliquots for protein determination
before the addition of BME- or DTT-containing
buffers. Once samples are dissolved in either Urea
Sample Buffer or SDS Boiling Buffer containing BME
or DTT their protein concentrations cannot be
measured. If samples are cloudy,
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dissolve them completely by boiling with SDS minus
BME before performing the BCA assay. Please be
sure to let us know if BME or DTT have been added
to the samples when mailing. Note: if you are using
the Bradford method SDS can’t be used because >
0.1% SDS interferes with this assay.
VI. Protein Precipitation Methods
Precipitation techniques are commonly employed to
separate sample proteins from interfering contaminants such as salts, detergents, nucleic acids, lipids
and charged sugars, and to concentrate proteins
without concentrating salt. Proteins in samples
containing high salt require either dialysis or precipitation, for example those scheduled for silver
staining with a protein concentration <1 mg/ml and
salt concentration > 200 mM, and samples
scheduled for Coomassie blue staining which have a
protein concentration < 4 mg/ml and salt >200 mM.
Depending on composition one technique may be
more suitable for your sample than another. Note
that precipitation can alter the protein profile of a
sample and should be avoided if possible.
A. Ethanol Precipitation is a simple method for
removing sample contaminants. Suspend one part
lysed or disrupted sample in nine parts absolute
ethanol in an Eppendorf tube and vortex. Allow the
suspension to sit at –80oC for at least 2 hours.
Microfuge for 30 minutes at 12-14,000 rpm in a
centrifuge cooled to 15-20°C (colder will bring down
large SDS pellet). Carefully remove the ethanol from
the samples and air-dry them for a short time to
remove residual ethanol.
B. Trichloroacetic acid (TCA) in acetone is
another common method used to precipitate proteins
during sample preparation for 2-D electrophoresis
and is more effective than either TCA or acetone
alone at removing starch and sugars [23].
Suspend lysed or disrupted sample in 10% TCA in
acetone with either 0.07% 2-mercaptoethanol or 20
mM DTT. Precipitate proteins for at least 45 minutes
at -20°C. Pellet proteins by centrifugation at
maximum rpm, 4°C, for 20 minutes. Wash the pellet
twice with cold acetone containing either 0.07% 2mercaptoethanol or 20 mM DTT by centrifuging at
maximum rpm, 4°C, for 5 minutes. Remove residual
acetone by air-drying.
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C. Precipitation with ammonium acetate in
methanol following phenol extraction [24] is
useful with plant samples containing high levels of
interfering substances. Begin by combining 100 ul
sample with 150 ul liquid phenol (best quality by
adding water to crystalline phenol). Add 10 ul of
10% SDS and 10 ul BME, vortex. Centrifuge for one
min at maximum rpm’s. Following centrifugation
remove upper phenol phase. Proteins are
precipitated from the phenol phase with 0.1 M
ammonium acetate in methanol. Pellet the
precipitated proteins with centrifugation. The pellet
is then washed several times with ammonium
acetate in methanol. Remove ammonium acetate
and allow any residual to evaporate.
D. Removal of lipid contaminants via protein
precipitation with chloroform/methanol [25]
Transfer the sample to a 1.5 ml microcentrifuge tube
and adjust the volume to 100 ul by lyophilization or
adding water. Add 400 ul of methanol and vortex.
Centrifuge the tube for 10 seconds at 9000xg, add
100 ul of chloroform and vortex. Centrifuge the
sample for ten seconds, add 300 ul water and vortex
vigorously to mix. Centrifuge the sample at 9000xg
for three minutes at 4°C. At this point the liquid
should have separated into two phases. If not, add
an additional 100 ul of chloroform and centrifuge
again.
Aspirate the upper phase with a narrow gauge
hypodermic needle or a pipetteman with a narrow
tip. Transfer sample to a fresh microcentrifuge tube.
Be careful not to disturb the interphase, the protein is
present in the interphase at this point. It is advisable
to leave 10-20 ul of upper phase behind. Add 300 ul
of methanol to the lower phase in the original tube
and vortex. Centrifuge at 14,000xg for ten minutes at
4°C. The protein should have formed a pellet. With
small amounts of protein the pellet may be rough
and smeared along the side of the tube. Transfer
the supernatant and air-dry or redissolved the pellet.
VII. Determining Protein Bound Dpm
Samples from radiolabeled cultured cells often have
protein concentrations too low to measure. However,
loading equal protein-bound dpm usually gives good
results as long as protein-bound dpm are distinguished from free dpm. For measurement purposes,
the protein-bound radioactivity may be separated
from free counts by TCA precipitation.
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The following method is derived from Garrels [8]:
For the TCA precipitation reserve 6 ul of each
sample for duplicate (3 μl each) measurements.
Prepare a bovine serum albumin (BSA) carrier
solution containing, per ml, 0.2 mg BSA and 1 mg
methionine to which urea sample buffer and
Coomassie blue have been added. For 110 ml of
carrier solution dissolve 20 mg BSA and 100 mg
methionine in 100mL water. Add 10 ml of Urea
Sample Buffer and 0.5 ml of a 0.05% Coomassie
blue stock solution. The stock solution contains 50
mg Coomassie brilliant blue R250 per 100 ml
distilled water. Coomassie blue is added so that the
pellet is easily visualized when aspirating the
supernatant.
Add 3 ul of sample in duplicate to 300 ul aliquots of
ice-cold BSA carrier solution in an uncolored 1.5 ml
microcentrifuge tube. Next, add 150 ul of ice cold
50% TCA and allow the tube to stand on ice for 5
min. Microfuge for 2 min and aspirate the
supernatant. Wash the pellet once by adding 300 ul
of ice cold 10% TCA, re-microfuge for 2 min, and
aspirate the supernatant.
Add 100 μl of Soluene 350 (Packard Instruments)
and vortex frequently at room temperature until the
pellet is dissolved; this may take up to one hour.
Place the open tube in a large scintillation vial with
10 ml of Ultima Gold scintillation fluid (Packard
Instruments). Mix thoroughly and determine protein
bound dpm per 3 ul of sample by scintillation
counting.
In some cases, knowing the specific activity (dpm/μ
g protein) is useful. Sample specific activity is
calculated by dividing the protein-bound dpm/μl by
the protein concentration. Optimal specific activities
for total cell lysates in dpm/μg protein are: 35S,
40,000; 14C, 40,000; 3H, 100,000; 32P, 20,000; and
125
I, 20,000.
VIII. Radiolabeling Cells and Tissues
For a review of the pros and cons of radiolabeling
experiments including a discussion of equilibrium,
pulse and pulse-chase formats, see Dunbar [26]
p103-116. For more detailed radiolabeling protocols
for cultured cells, yeast and bacteria see Spector
[27]. For a specific example see Anri et. al. [28]. In
addition, many specific protocols may be found in
the literature.
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Examples of cell labeling with 35S-methionine and
32
P orthophosphate are given below.
Labeling media: We define labeling media to be
serum free media lacking the cold amino acid
corresponding to the radiolabeled amino acid. For
example, cold methionine would be omitted when
radiolabeling with 35S-methionine to increase the
efficiency of incorporation. At the start of the
experiment, the labeling media should contain 250–
1000 μCi of the radiolabeled amino acid/ml. Be sure
to treat control samples and test samples identically.
Use the smallest amount of radioactive labeling
media possible to culture cells. Make sure it is at the
proper temperature before adding it to the dish.
Avoid starving the cells, changing the pH, or
changing the osmolarity prior to labeling; such
changes may produce artifacts.
A.
35
S-methionine labeling - cultured cells
Plated cells: Pick cells that have formed a confluent
layer over the surface of the tissue culture dish.
Rinse the cells with warmed media minus the
labeling amino acid. Incubate the cells for 20 min at
37°C in the same prewarmed medium to deplete the
intracellular pools. Remove this medium by
aspiration and add a minimal amount of labeling
media containing 35S-methionine. For example, to a
35 mm dish add 250 μl of labeling media containing
250–500 μCi radiolabel per 40,000–400,000 cells/
cm2. The cell layer should be cultured in labeling
media for 2–12 hrs. You may need to run a pilot
experiment to determine labeling times; four hrs is a
good starting point.
Suspended cells: Remove enough cell fluid to
harvest 2–4 million cells; gently spin down the cells
at 400 x g for 5 min and remove the culture medium.
Wash the cells by resuspending in prewarmed
labeling medium minus methionine, re-centrifuge
and remove the buffer. Resuspend the cells again in
labeling medium without methionine and incubate for
20 min at 37°C to deplete the intracellular pools. Repellet the cells and resuspend in 0.5 ml of labeling
media containing 250–500 uCi of 35S-methionine.
Incubate the cells in the media for 2–6 hours, pellet
the cells and discard the supernatant into radioactive
waste. Wash the cells twice in PBS before lysing.
www.kendricklabs.com
B. 32P labeling of cultured cells
First, consider how you will minimize body exposure to
radiation and avoid contamination of laboratory areas.
Obtain 32P as orthophosphate (32P04-3) in an aqueous,
HCI-free and carrier-free solution. Reduce the labeling
media phosphate concentration to 50 uM or lower and
add 500–1000 uCi of 32P/0.5 ml of media for
suspended cells or 250–500 uCi of 32P/ml for plated
cells.
Limit the labeling time to 5 hrs or less to reduce 32P
incorporation into DNA and phospholipid. After
radiolabeling, rinse the cells in tris buffered saline (150
mM NaCl, 50 mM Tris-HCI, pH 7.5, 0.1 mM EDTA).
Prepare these samples as described for 35S samples of
a similar type with the following exception: do not treat
with 10× Nuclease Stock solution. If you do, DNA and
RNA fragments will isoelectric focus and create
background problems. Instead, after adding SDS
Boiling Buffer (or Urea Sample Buffer), centrifuge at
100,000–200-000 × g for 1–2 hrs to remove nucleic
acids [26].
IX. Mailing Samples
6. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia,
A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K.,
Goeke, N.M., Olson, B.J., Klenk, D.C., Anal Biochem,
1985. 150: 76-85.
7. Lelivelt, M.J.,Kawula, T.H., J Bacteriol, 1995. 177:
4900-7.
8. Garrels, J., Meth. Enzymol, 1983. 100: 411-23.
9. Jeffcoate, S.L.,White, N., J Clin Endocrinol Metab,
1974. 38: 155-7.
10. Markwardt, F., Walsmann, P., Sturzebecher, J.,
Landmann, H., Wagner, G., Pharmazie, 1973. 28: 32630.
11. Aoyagi, T., Takeuchi, T., Matsuzaki, A.,
Kawamura, K., Kondo, S., J Antibiot (Tokyo), 1969. 22:
283-6.
12. Oakley, B.R., Kirsch, D.R., Morris, N.R., Anal
Biochem, 1980. 105: 361-3.
13. O'Connell, K.L.,Stults, J.T., Electrophoresis, 1997.
18: 349-59.
14. Bonner, W.M.,Laskey, R.A., Eur J Biochem, 1974.
46: 83-8.
15. Anderson, N.L.,Hickman, B.J., Anal Biochem,
1979. 93: 312-20.
16. Zhang, Z., Izaguirre, G., Lin, S., Lee, H., Schaefer,
E., Haimovich, B., Mol Biol Cell, 2004. 15: 4234-47.
17. Darbre, A., Practical protein chemistry : a
handbook. 1986, Chichester Sussex ; New York: Wiley.
xix, 620.
Fill out the sample ID form and store the samples
frozen at -70°C until mailing. Mail samples frozen in
Eppendorf tubes on at least 6 pounds of dry ice by
overnight mail. See www.kendricklabs.com “2D
Services” (drop down menu) “how to send samples”
for more details.
18. Jazwinski, S., Meth Enzymol, 1990.182: 154-74.
If buffers are unavailable, send the proper amount of
protein aliquoted or lyophilized in a 1.5 ml Eppendorf
tube. We will add the appropriate buffer as indicated on
the sample ID form. There is no charge for this as long
as the amount of standard buffer to be added is clear.
22. Bradford, M., Anal Biochem, 1976. 72: 248-54.
X. Reference List
1.Donat, T.L., Sakr, W., Lehr, J.E., Pienta, K.J.,
Otolaryngol Head Neck Surg, 1996. 114: 387-93.
2. Anderson, N.G.,Anderson, N.L., Anal Biochem,
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3. O'Farrell, P.H., J Biol Chem, 1975. 250: 4007-21.
4. O'Farrell, P.Z., Goodman, H.M., O'Farrell, P.H.,
Cell, 1977. 12: 1133-41.
5. Burgess-Cassler, A., Johansen, J.J., Santek, D.A.,
Ide, J.R., Kendrick, N.C., Clin Chem, 1989. 35: 2297304.
8
800-462-3417
19. Brown, K., Gerstberger, S., Carlson, L., Franzoso,
G., Siebenlist, U., Science, 1995. 267: 1485-8.
20. Chen, X., Ding, Y., Liu, C.G., Mikhail, S., Yang,
C.S., Carcinogenesis, 2002. 23: 123-30.
21. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall,
R.J., J Biol Chem, 1951. 193: 265-75.
23. Pang, L., Fryksdale, B.G., Chow, N., Wong, D.L.,
Gaertner, A.L., Miller, B.S., Electrophoresis, 2003. 24:
3484-92.
24. Meyer, Y., Grosset, J., Chartier, Y., Cleyet-Marel,
J.C., Electrophoresis, 1988. 9: 704-12.
25. Wessel, D.,Flugge, U.I., Anal Biochem, 1984. 138:
141-3.
26. Dunbar, B.S., 2-D electrophoresis, and
immunological techniques. 1987, New York: Plenum
Press. xvi, 372 , [2] of plates.
27. Spector, D.L., Goldman, R.D., Leinwand, L.A.,
Cells : a laboratory manual. 1998, Cold Spring Harbor,
NY: Cold Spring Harbor Lab Press. 3 v.
28. Amri, H., Drieu, K., Papadopoulos, V.,
Endocrinology, 1997. 138: 5415-26.