Desalting, Concentration, and Buffer UNIT 4.

4
Exchange by Dialysis and Ultraltration
Many steps in protein purication result in the introduction of low-molecular-weight
materials, such as (NH4 )2 SO4 , that exceed levels appropriate for subsequent procedures.
In cases where simple dilution cannot sufciently reduce the concentration of these com-
pounds, dialysis or dialtration (dialysis by ultraltration at constant volume) are options
which should be considered. Ultraltration is also convenient for concentrating protein
samples without concomitantly concentrating low-molecular-weight substances. This is
achieved by forcing water and dissolved low-molecular-weight compounds through a
semipermeable membrane under pressure; the high-molecular-weight protein compo-
nents are retained behind the membrane. Alternatively, ultraltration can be carried out
via a rapidly recirculating ow of solution over a semipermeable membrane (tangential-
ow ultraltration). Dialysis and ultraltration are also effective methods for buffer
exchangei.e., the replacement of a buffer used for a previous procedure with a dif-
ferent buffer appropriate for a subsequent one.

Gel ltration (UNIT 8.3) can also be used for desalting, but because this purication tech-
nique normally employs a column containing a differentially porous gel matrix rather
than a semipermeable membrane, it is less readily adapted to large sample volumes or
to situations where multiple samples are to be desalted. However, despite the availability
of membranes with a wide range of porosities, dialysis and ultraltration are generally
less efcient than gel ltration for separation of proteins on the basis of size differences,
except where the difference in sizes is quite large (i.e., by a factor of 10; see Background
Information).

This unit includes a variety of dialysis and ultraltration techniques that can be
used for desalting, concentration, or buffer exchange. The rst method (see Basic
Protocol 1) describes standard dialysis by diffusion across cellulose tubing, a technique
used for desalting or buffer exchange. The second (see Basic Protocol 2) describes ul-
traltration under pressure, which can be used either for concentrating protein or, where
the sample volume is replenished with a desired buffer, for desalting/buffer exchange
(i.e., dialtration). In addition a method utilizing ultraltration by tangential ow, which
serves the same purposes as ultraltration under pressure but is better suited to handling
large volumes of solution, is also described (see Alternate Protocol 1). Finally, an ultral-
tration technique employing centrifugal microconcentrators, which allow concentration
of small volumes of solution, is given (see Alternate Protocol 2).

NOTE: Additional information on standard dialysis using membrane diffusion is provided

in APPENDIX 3B.

DESALTING AND BUFFER EXCHANGE BY DIALYSIS THROUGH BASIC

REGENERATED CELLULOSE TUBING PROTOCOL 1
When the sample volume is between 0.2 and 200 ml, dialysis can be accomplished by
placing the sample in standard dialysis tubing immersed in a buffer of the desired ionic
composition and strength, with continuous stirring of the buffer. For larger volumes,
multiple dialysis bags may be used, but desalting or buffer exchange on a large scale
is more rapidly achieved by tangential-ow dialtration (see Alternate Protocol 1). A
single dialysis bag formed with large diameter tubing, when compared with multiple
Extraction,
thinner diameter bags holding the same total volume, is less efcient due to the longer Stabilization,
and
Concentration
Contributed by Allen T. Phillips and Mark W. Signs 4.4.1
Current Protocols in Protein Science (2004) 4.4.1-4.4.15
Copyright 
C 2004 by John Wiley & Sons, Inc.
Supplement 38
 diffusion path for materials inside the bag. Because the exposure time of the sample to
the dialysis buffer is relatively long, the composition, pH, and ionic strength of the buffer
should be dictated by considerations of protein stability; when large volumes are involved,
buffer price may be a factor as well. Extremely small samples (10 to 100 l) are best
dialyzed in specially designed units such as the Spectra/Por Micro Dispo/Dialyzer with
an appropriately chosen membrane, or by centrifugal ultralters used in a dialtration
mode (see Alternate Protocol 2).

Materials
Dialysis tubing: regenerated cellulose, molecular weight cut-off (MWCO) 12,000
(e.g., Spectra/Por Type 4, Spectrum) for general protein use or MWCO 3500
(e.g., Spectra/Por Type 3) for retention of small proteins, stored at 4 C in a
tightly closed plastic bag
50% (v/v) ethanol in large wash bottle
10 mM EDTA, pH 8.0
0.05 M NaHCO3
Protein solution (clarify by centrifugation if necessary)
Appropriate dialysis buffer
Large wash bottle
Weighted and unweighted tubing closures (Spectrum)
Small plastic funnel
Glass marble or glass beads (optional): rinse thoroughly with dialysis buffer before
use
Conductivity meter and probe (optional)

Prepare the dialysis tubing

1. Select tubing of appropriate MWCO that has a width such that the sample can be
contained in one or more sections 30 cm in length. Cut the dry tubing to the desired
length, allowing an additional 10 cm total length for closing off the ends.
Handle tubing only with powder-free gloves to avoid getting skin oils on membrane.
Longer sections may be used, provided they can be completely immersed in the dialysis
buffer without bending the lled tubing (see Table 4.4.1).

2. Immerse the sections of tubing in distilled water and rub each between two ngers
until the layers separate. Using a large wash bottle containing 50% ethanol, rinse
the inside and outside of each section thoroughly.
The ethanol rinse serves to remove glycerol (used as a humectant) and residual suldes
(from the manufacturing process); the remaining traces will be removed in the next two
steps.

Table 4.4.1 Approximate Capacities for Dialysis Tubing of Various Diameters

Flat width (mm) Inated diameter (mm) Volume/length (ml/cm)

10 6.4 0.3
18 11.5 1.0
25 16 2.0
Desalting,
Concentration, 45 29 6.4
and Buffer
Exchange by 54 34 9.3
Dialysis and
Ultraltration 75 48 18

4.4.2
Supplement 38 Current Protocols in Protein Science
 3. Mix 400 ml of 10 mM EDTA (pH 8.0) and 400 ml of 0.05 M NaHCO3 in a 1-liter
beaker. Transfer the wetted and washed tubing to this beaker and stir 30 min using
a magnetic stirrer.
4. Decant the EDTA/NaHCO3 solution and replace with 800 ml distilled water. Stir
10 min on the magnetic stirrer, decant the water, and repeat the water wash. Transfer
the tubing to fresh distilled water.
If not to be used promptly, the tubing should be stored covered at 4 C in water containing
0.1% (w/v) sodium azide. Once wetted, the membranes must not be allowed to dry out.
Boiling dialysis tubing is not recommended for use in protein work. For more vigorous
removal of glycerol and sulde in situations where these impurities could be troublesome,
the tubing may be immersed in 1 liter of 50% ethanol and heated 1 hr at 50 C, instead of
being rinsed as in step 2. Regenerated cellulose tubing may be autoclaved, but this alters
the MWCO. Spectra/Por Type 7 membranes are precleaned and supplied wet; they are
available in a variety of diameters and MWCO values. For critical applications, tubing
manufactured by an alternate process and containing no glycerol, suldes, or heavy
metals is available in MWCO values between 3,500 and 60,000 (Spectra/Por Biotech
Regenerated Cellulose Membranes, Spectrum). Because this tubing is available mainly
in narrow at widths (4 to 16 mm), it is most suitable for smaller samples (0.25 to 20 ml).
Also available is cellulose ester membrane tubing (Spectra/Por Biotech Cellulose Ester
dialysis membrane, Spectrum), which requires no pretreatment and comes in MWCO
values from 100 to 300,000 Da. This material is less tolerant of organic solvent presence
than are regenerated cellulose membranes, however.

Dialyze the sample

5. Close one end of a washed section of tubing rmly with a weighted tubing closure,
being sure that the closure is longer than the at width of the tubing. Squeeze the
tubing between two ngers to remove excess water from inside.
Alternatively, the tubing section may be closed by tying two simple overhand knots
near one end. If membranes other than regenerated cellulose types are used, follow the
manufacturers suggestion on closure methods to avoid damage.

6. Slip the open end of the tubing section over the narrow end of a small plastic funnel,
then add the protein sample through the funnel. When sample addition is complete,
remove the funnel and work the sample down the tubing by squeezing with the
ngers to eliminate air space.
For small samples (10 ml) addition by a pipet may be more convenient.

7. If a weighted tubing closure was not used in step 5, place a well-rinsed glass marble
or several large glass beads in the bag to serve as weight, thus keeping the entire
sample immersed during dialysis.
8. Clamp the end shut with a nonweighted tubing closure (or knot the tubing carefully
twice; see step 5) and trim off excess tubing. Check for leakage at both ends by
applying moderate pressure to the sample area.
Excessive air space will lead to dilution of the sample by buffer that enters and displaces
air during dialysis; however, extra space (5% to 20% of the total bag volume) should be
allowed if the sample has a very high salt concentration, as it may be necessary to permit
an increase in volume to prevent rupture of the bag. Moreover, a moderately sized air
bubble at one end can be useful for mixing of contents by simple inversion periodically
to reduce concentration polarization (see Critical Parameters). If the sample is very
viscous, it should be diluted with the buffer against which it will be dialyzed to facilitate
outward diffusion.
Extraction,
9. Place the lled dialysis bag in a beaker or large at-bottomed plastic vessel con- Stabilization,
taining at least 10 vol. dialysis buffer, relative to the sample volume, at the desired and
Concentration

4.4.3
Current Protocols in Protein Science Supplement 38
 temperature, making sure the bag is completely immersed. Stir the dialysis buffer
continuously with a magnetic stirrer.
The volume of dialysis buffer should be at least ten times the volume of the samples to be
dialyzed, and preferably much larger. See Background Information for a discussion of
dialysis buffer volume versus sample volume and using a large volume of dialysis buffer
or changing smaller volumes several times.
Continual stirring serves to maximize the difference in salt concentration between the
interior and immediate exterior of the dialysis bag. A larger initial buffer-to-sample ratio
(100:1) will speed attainment of the desired nal ionic strength with fewer changes
of dialysis buffer. Although the diffusion rate is slightly reduced in the cold, almost all
protein dialysis is conducted at 4 C to enhance protein stability.

10. When desalting is desired (as opposed to simply a buffer change), perform the
following procedure:

a. Monitor the progress of the dialysis by measuring the conductivity of the

dialysis buffer with a conductivity meter if at all possible.
b. Replace the used dialysis buffer with an equal volume of fresh buffer when
the increase in conductivity slows down (indicating approach to equilibrium
and a drop in the efciency of the dialysis).
c. Continue replacing the dialysis buffer in this way until the conductivity of the
buffer remains essentially unchanged after stirring for 2 hr.
If a conductivity meter is not available, the buffer should be changed at least twice,
allowing 6 hr between changes to permit the system to approach equilibrium.
Conductivity measurements are particularly useful when large amounts of salt are to be
removed and when the buffer-to-sample ratio is 100:1. In such cases, several replace-
ments of dialysis buffer will be necessary to achieve the desired nal ionic strength; con-
ductivity measurements permit dialysis to be completed in minimum time by eliminating
guesswork regarding when the system is approaching equilibrium. When a conductivity
meter is not used and large samples (or tubing diameters >20 mm) are involved, it may
be necessary to extend the time between buffer changes to 8 to 12 hr to insure that
equilibrium is attained.

11. Remove the dialysis bag from the buffer and rinse the exterior with distilled water.
While holding the bag just below the upper closure (or knot), remove that closure
(or cut off the knot).
12. Carefully tip the bag and pour the dialyzed sample into an appropriate container.
Conrm that the desired salt concentration was achieved by comparing conductivity
measurements of the dialyzed sample and unused dialysis buffer.
If the bag is extremely taut as a result of buffer (water) inltration, do not cut the upper
knot; instead put one end of the tubing into a beaker large enough to hold the entire
sample, then puncture the tubing near the lower knot with a long needle and allow the
sample to drain directly into the beaker.

BASIC CONCENTRATION OR DIALYSIS BY ULTRAFILTRATION THROUGH

PROTOCOL 2 ASYMMETRIC MEMBRANE DISKS UNDER PRESSURE
Protein samples at an initial concentration of 20 mg/ml can easily be reduced in volume
using an ultraltration system and membrane, as in the protocol described below. The
stirred-cell system described in this protocolin which pressure from nitrogen gas
Desalting,
Concentration, forces molecules which are much smaller than the cutoff (MWCO) through the poresis
and Buffer available with a variety of chamber sizes and membrane properties. Because most salts
Exchange by pass through the membrane along with water, the sample volume is reduced without
Dialysis and
Ultraltration

4.4.4
Supplement 38 Current Protocols in Protein Science
a signicant change in its surrounding buffer composition, an obvious advantage over
lyophilization.

Ultraltration can also be used to remove or exchange salts without changing the pro-
tein concentration. This process, called dialtration, resembles dialysis but is driven by
the rate of ultraltration (see Critical Parameters and Troubleshooting) as opposed to a
concentration gradient. Dialtration is carried out as in the concentration protocol here,
except that continuous or periodic additions of buffer of the desired nal pH, concentra-
tion, and composition are made to compensate for changes in protein concentration that
would otherwise occur as a result of volume reduction.

The pressurized systems discussed here are sometimes referred to as dead-end ltration
systems, in contrast to tangential or cross-ow systems described elsewhere in the unit
(see Alternate Protocol 1). The latter are usually more complex but handle larger volumes
more easily and are less susceptible to concentration polarization (see Critical Parameters
and Troubleshooting).

While the overall principle is similar to that described in this protocol, the Vivacell 70
concentrator (Sartorius AG) can use either gas pressure, centrifugal force, or both to
drive the liquid through its polyethersulfone membrane. Pressurization of the sample
reservoir is achieved and then the vessel is sealed, thus eliminating connections between
the pressure source and the sample container. This allows the vessel to be placed on a
laboratory shaker as a way to reduce concentration polarization, or placed in a centrifuge
rotor and spun to accelerate ultraltration. A built-in dead stop prevents concentration to
dryness. Membranes are reusuable and come in several MWCO values.

Materials
Protein solution (clarify by centrifugation if necessary)
N2 gas cylinder and two-stage pressure regulator capable of controlling delivery up
to 100 psi
5% ethanol
Ultraltration membrane (YM10, Amicon; Ultracel PLGC, Millipore; or PM10,
Amicon) with MWCO >10,000 and diameter to t stirred cell
Ultraltration unit, stirred-cell type (Amicon Series 8000, Millipore) with:
Glass funnel with stem diameter to t through cell ll hole (optional; used in
certain cell models)
Ultraltrate collection container (Grifn beaker with graduations)
Tubing for connection to N2 source
Pressurized reservoir (optional; for use when sample size is large and protein is
dilute, or when dialtration is performed with constant readdition of new buffer;
e.g., RG5 Fiberglass Reservoir, Amicon)
NOTE: Handle the membrane only with powder-free gloves and hold by the edge to avoid
scratching the smooth, thin skin surface.

1. Rinse a new ultraltration membrane with sufcient distilled water to wet it thor-
oughly.
Some ultraltration membranes can be used more than once. The Millipore PLGC
(MWCO 10,000) or PLTK (MWCO 30,000) cellulose acetate membranes are intended
mainly for single use. Many other suppliers indicate that their membranes can be reused
until clogged or damaged. Such membranes may be more economical, but only if the
user makes sure that the previously used membrane is undamaged and properly cleaned. Extraction,
Amicon YM10 or PM10 membranes can be cleaned by soaking in 0.01 M SDS solution Stabilization,
or 1% (w/v) Terg-A-Zyme (VWR) for several hours; the membranes are then thoroughly and
Concentration
rinsed with distilled water before reuse. Used, cleaned membranes should be stored
4.4.5
Current Protocols in Protein Science Supplement 38
 covered with 5% ethanol in a closed container at 4 C. For membranes other than the
ones mentioned, the manufacturers instructions must be consulted for a suitable clean-
ing procedure.

2. Load the membrane into the stirred cell of the ultraltration unit with the skin or
glossy side up. Complete assembly of the ultraltration system, taking special care
to ensure that O ring seals (if used) are not nicked and are properly placed on the
membrane disk to prevent leakage when pressure is applied.
The size and thus the membrane diameter of the ultraltration cell should be as large as
practical for the amount of sample to be concentrated; a larger cell reduces the necessity
to rell the unit and provides the largest ltration area, speeding the concentration
process. Cells are commonly available in sizes ranging from 10 to 400 ml. While YM10 or
Ultracel PLGC membranes are recommended for concentrating dilute solutions because
of their minimal protein adsorption, the PM10 polyethersulfone membrane operates more
rapidly with high concentration samples like plasma.

3. Fill stirred cell with sample to the maximum operating volume, either by pouring
the sample directly through the ll hole or, when using systems where the ll hole
is small, by using the glass funnel provided with the cell.
If a funnel is used, contact between the funnel tip and membrane surface should be
avoided.

4. Replace the ll-hole cover and check that the pressure-release valve (often an inte-
gral part of the ll-hole cover) is in the closed position.
5. Place the unit on a magnetic stirrer and stir at the maximum rate that does not
generate foam or a sizeable vortex at the surface of the solution. Direct the outlet
tubing to the ultraltrate collection container.
Because of protein stability requirements, most ultraltrations are conducted in the cold.
It is advisable to check the surface temperature of the magnetic stirrer periodically; if
it is warm, a Styrofoam sheet 0.5 in. thick should be placed between the stirrer surface
and the cell bottom.

6. Connect the cell to the nitrogen gas cylinder with two-stage pressure regulator and
pressurize to 55 psi.
Flow of liquid into the outlet tubing should quickly become evident. The system should
be examined for appearance of liquid at the base of the cell where the cylinder body
contacts the O ring; this indicates a leak around the membrane, in which case the unit
should be depressurized and its assembly examined.
Lower or higher pressures may be needed depending on the membrane type and MWCO
value, but pressure should not exceed 70 psi in most stirred cells.

7. Monitor the ow rate and ultraltrate volume until the desired level of concentration
has been achieved. If the sample volume is many times the cell capacity, use a pres-
surized sample reservoir (if available) to add new sample continuously; otherwise,
add the sample periodically as described in step 3.
If the total sample volume exceeds the cell capacity, the cell should be relled when the
volume has decreased by one-third. Sample additions are then continued until all of the
sample has been added to the cell.
At a constant temperature and pressure, the ow rate is a function of the lter area and the
degree to which concentration polarization can be avoided (see Critical Parameters and
Desalting,
Concentration, Troubleshooting). Vigorous stirring is needed to minimize concentration polarization of
and Buffer protein at the membrane surface. Buildup of protein on the surface will result in slow
Exchange by ltration, even when the protein concentration of the sample is relatively low. Filtration
Dialysis and rates at 4o C are often only one-half those seen at 25 C, because of the inuence of
Ultraltration
viscosity.
4.4.6
Supplement 38 Current Protocols in Protein Science
 Do not concentrate the sample to the point where it is no longer in contact with the
stir bar, as lack of stirring will lead to severe concentration polarization in all but the
most dilute samples. Overconcentration makes sample recovery difcult and may require
readdition of buffer to wash the membrane, thereby adding to the volume.

8. Turn off the stirrer and the nitrogen ow to the cell. Slowly release the internal cell
pressure, then turn the stirrer back on and allow stirring to continue for 2 min to
resuspend protein in the gel layer.
9. Turn the stirrer off and remove the concentrated sample by pouring or pipetting
through the ll hole.
If a pipet is used and the membrane is of the reusable type, care should be taken not to
touch the pipet to the membrane surface. An alternative means of removing the sample
is to unclamp the cell and pour out the sample, after checking that the body of the unit
is rmly screwed into the base.

10. If the membrane is of the reusable type, hold it by the edges and rinse well with
distilled water. Proceed as soon as possible with cleaning (see step 1), or follow
the manufacturers suggested cleaning procedure, keeping the membrane wet at all
times.
11. Store the cleaned lter indenitely at 4 C in a closed vessel containing 5% ethanol.

DIAFILTRATION OR CONCENTRATION BY TANGENTIAL-FLOW ALTERNATE

ULTRAFILTRATION PROTOCOL 1
The most rapid method for dialyzing large samples is the use of ultraltration membranes
fabricated into hollow bers or spiral-wound layers, or, as in this protocol, in the form
of at plate cassettes. The sample is allowed to ow tangentially over these membranes
with recirculation. The semipermeable nature of the membrane then permits water and
materials with molecular weights that are substantially below the MWCO value to cross
through the membrane surface relatively unimpeded; retained materials, including the
proteins of interest, are returned to the sample reservoir. To use this technique for di-
altration (as opposed to concentration), fresh dialysis buffer of the desired composition
should be added to the sample reservoir in a volume equal to that of the ultraltrate col-
lected, so that the concentration of macromolecular species remains unchanged. Removal
of microsolutes to which the membrane is completely permeable can be estimated from
the volume of ultraltrate (or dialtrate, in this case) collected. Collection of an amount
of dialtrate equal to 3 vol. of original sample will remove 95% of the starting solute to
which the membrane is permeable, while a dialtrate of 5 vol. will correspond to a 99%
separation.

The same process is used to concentrate samples, except that the retentate volume is
allowed to decrease until the desired degree of concentration is reached. Maintaining
a rapid ow over the membrane with a moderate transmembrane pressure minimizes
concentration polarization and keeps the ltration rate high even as protein concentration
in the sample increases. It is the reduction in concentration polarization as a result of
the tangential ow across the membrane surface that most distinguishes this type of
ultraltration from the previously discussed dead-end ltration method.

The procedure described here is based on the Pellicon XL 50 ultraltration device that
employs a at-plate membrane and a peristaltic pump. It has a membrane lter area of
50 cm2 and is designed for initial sample sizes from 50 ml to 1 liter for a single XL 50
Extraction,
unit; larger volumes generally require more membrane area, achieved by placing multiple Stabilization,
XL 50 units in series, in order to accomplish dialtration or concentration in conveniently and
Concentration
short times (6 hr). Alternatively, the Pellicon 2 (Millipore) cassette tangential ow
4.4.7
Current Protocols in Protein Science Supplement 38
 ltration device, with a minimum working volume of 250 ml, can be readily expanded
to areas up to 0.3 m2 and is directly scalable from the XL 50 unit. Equivalent, hollow
ber units include the Spectrum MidiKros for volumes of 10 to 200 ml and the Spectrum
MiniKros system for volumes of 300 ml to 100 liters. All units are designed to accept
regenerated cellulose or polyethersulfone membranes in a variety of MWCO values.
The former membrane material is recommended for dilute protein solutions since it has
one-half or less the protein adsorption noted with polyethersulfone membranes.

Materials
Dialysis buffer
Protein sample
0.2% (w/v) Terg-A-Zyme detergent (e.g., VWR), 40 C
0.05% (w/v) sodium azide
Pellicon XL 50 (Millipore) equipped with a membrane lter plate of cellulose
(PLCGC, MWCO 10,000 or PLCTK, MWCO 30,000) or polyethersulfone
(Biomax 30, MWCO 30,000)
Luer-to-barb connectors
4-mm-i.d. tubing (Tygon or silicone)
Sample and retentate collection reservoirs: containers capable of holding entire
sample
Hose clamps, adjustable with screws
Permeate collection vessel: Erlenmeyer ask larger than the sample volume
Variable speed peristaltic pump equipped with pump head of 480 ml/min rated
capacity (equivalent to that supplied with the Millipore Labscale TFF system)
10-ml syringe

Prepare the system

1. Select a lter plate of appropriate MWCO and chemical composition.
Each XL 50 comes with a specic membrane and thus several units with different mem-
branes may be needed to cover all applications.
Filtration rates are higher with 30,000-Da-MWCO membranes, but the large volumes
to be ltered in dialtration may lead to some loss of proteins with molecular weight
<50,000 using such membranes. Membranes with MWCOs of 5,000 Da are also avail-
able, but these lter more slowly.

2. Remove the luer caps and replace with luer-to-barb connectors. Attach 4-mm-i.d.
tubing of the type recommended for use with the peristalitic pump to the feed inlet
and run it through the pump to the sample reservoir.
Four luer-to-barb connectors are required.

3. Attach tubing to the retentate outlet and bring it to a retentate collection reservoir.
4. Attach a hose clamp, adjustable with screws, on this section of tubing to permit the
transmembrane pressure to be increased if necessary but under no circumstances
close this clamp fully during operation.
Three hose clamps are required for this procedure, including the one used in this step.

Desalting,
5. Attach tubing from the permeate 2 outlet, or both permeate 1 and 2 outlets, to a
Concentration, permeate collection vessel. Provide each section of outlet tubing with a hose clamp.
and Buffer
Exchange by A Y-shaped tube connector can be used to direct the output of both permeate exit ports
Dialysis and to a single piece of tubing, although Millipores instruction guide does not mention use
Ultraltration
of the permeate 1 outlet.
4.4.8
Supplement 38 Current Protocols in Protein Science
 6. Place dialysis buffer in the sample reservoir and slowly bring the variable-speed
peristaltic pump to a mid-range operating speed.
After air is purged from the permeate side of the membrane, both permeate outlets can
be used. A cross-ow rate for retentate, measured by timing collection in a graduated
cylinder, should be 30 to 50 ml per min.

7. Run the pump until 200 ml dialysis buffer has circulated through and purged the
lter plates and all tubing lines. Stop the pump and discard the contents of the
retentate collection reservoir and permeate collection vessel.
New XL 50 units contain glycerol and preservatives and need to be ushed out before
initial use; also storage solution from previous uses must be washed out. If buffer quantity
is limited, perform the initial wash with water, then switch to 50 ml buffer for a nal
ush. The unit can be operated in either a vertical or horizontal conguration but the
former facilitates removal of air from interior spaces.

Perform ultraltration
8. Fill the sample reservoir with the protein sample to be dialtered or concentrated.
Place the retentate-line tubing and feed-line tubing in the sample reservoir, then
place the reservoir on a stirrer and initiate active mixing.
9. Restart the pump and adjust the ow to 100% recirculation by clamping off the
permeate outlets. Measure the ow and readjust the pump speed as necessary until
the cross-ow is between 30 and 50 ml per min.
10. Open the permeate outlets and begin collection of the ultraltrate.
If ltrate ow is inadequate, some increase can be achieved by partially restricting the
retentate outlet line by adjusting the screw on the hose clamp, thereby increasing the
transmembrane pressure; however, reduced tangential ow can lead to greater concen-
tration polarization and ultimately a reduced dialysis or ultraltration rate. Increasing
the pump speed may provide greater cross-ow but risks exceeding the recommended
inlet pressure level for the tubing being used (usually 25 psi for silicone tubing with
4 mm i.d.); the XL 50 itself can handle a maximum transmembrane pressure of 40 psi.
Addition of another XL 50 unit doubles the membrane effective ltration surface area
and thus increases the permeate ow proportionally.

11a. For dialtration: Keep the sample reservoir lled as ltration proceeds by adding
fresh dialysis buffer to maintain the original volume. Continue adding fresh dialysis
buffer until the volume of permeate is three to ve times the volume of the original
sample.
Dialtration is most efcient when addition rate equals the permeate ow rate.

A permeate volume three to ve times the volume of the sample indicates that removal
of diffusible material is 95% to 99% complete.

11b. For concentration: Allow the sample volume to decrease until the appropriate new
volume is achieved.
12. Clamp off the permeate line(s) and stop the pump to terminate ltration. Carefully
remove tubing from the peristaltic pump.
13. To recover product from the XL 50 holdup volume (typically 4 ml plus any amount
in tubing), perform the following procedure:

a. Disconnect the permeate line and recap the port(s). Extraction,

Stabilization,
b. Elevate the retentate tubing and fully open the clamp on the retentate line to and
Concentration
allow sample to drain back into the feed reservoir.
4.4.9
Current Protocols in Protein Science Supplement 38
 c. Remove the retentate tubing and connector. Fill a 10-ml syringe with 4 to 5 ml
dialysis buffer and inject this into the retentate port to displace product held
inside the XL 50.
Clean the system
14. Clean the units membrane by reassembling the XL 50 as for regular use, then
pumping water through all lines to ush out remaining sample and buffer salts,
much as was done in steps 6 and 7.
15. Replace water with a 0.2% (w/v) Terg-A-Zyme detergent, 40 C, then move the
permeate and retentate tubing lines into the cleaning solution reservoir so as to
allow for total recirculation.
16. Pump the cleaning solution through the system for 30 to 60 min. Finally, repeat the
water ush for 30 min, discarding all permeate and retentate line output.
Alternative cleaning solutions include detergents such as 0.1% (w/v) SDS or 0.1% (v/v)
Triton X-100, both used at 40 C.

Store
17. Disconnect the retentate tubing from the XL 50 and remove the feed tubing from
the pump.
18. Fill a 10-ml syringe with 10 ml of 0.05% (w/v) sodium azide and slowly inject
into the retentate port.
19. When completed, remove the syringe and disconnect all tubing. Replace the units
luer caps and store indenitely at 4 to 25 C, both closed and at.

ALTERNATE MICROSCALE CONCENTRATION AND DESALTING WITH

PROTOCOL 2 CENTRIFUGAL ULTRAFILTERS
Sample preparation for analytical purposes and small-scale purication may require con-
centration and/or desalting of volumes between 0.2 and 20 ml. Stirred-cell assemblies
(see Basic Protocol 2), although able to deal with amounts that overlap this range, often
have unacceptably large internal volumes, making sample recovery difcult. Moreover,
as concentration proceeds, the risk of excessive volume reduction (even to dryness) in-
creases because ltration with stirred cells is typically not self-limiting.

The advent of commercially available, single-use ultraltration microconcentrators pow-

ered by centrifugal force has eliminated many of these problems. These are available with
a range of MWCO values in sizes suitable for handling volumes of 50 l to 2 ml (e.g.,
Microcon units from Amicon). Centrifugal ultralters are also produced in sizes that
will accommodate samples of 2 to 20 ml. Early models had limited membrane area and
minimal remixing which resulted in low rates of concentration as compared with stirred
cells, except for very dilute samples. However, recent designs have greatly reduced con-
centration polarization (e.g., Ultrafree and Centriprep units from Amicon); these mostly
involve improved methods of producing movement of liquid through the membrane via
centrifugal force.

Materials
Centrifuge microconcentrator units: e.g., Microcon-10, -30, or -50 (MWCO
10,000, 30,000, and 50,000 respectively; Amicon); or Ultrafree-MC with
Desalting,
Concentration, MWCO 5,000, 10,000, or 30,000 (Millipore)
and Buffer Microcentrifuge, 4 C, with either a xed-angle or swinging-bucket rotor, capable
Exchange by of providing forces up to 13,000 g
Dialysis and
Ultraltration

4.4.10
Supplement 38 Current Protocols in Protein Science
 1. Insert the lter of the centrifuge microconcentrator unit into the supplied centrifuge
tube. Pipet 500 l protein solution into the microconcentrator and seal with the
cap supplied by the manufacturer. Take special care to avoid touching the membrane
surface with the pipet tip.
2. When using a single concentrator or when multiple ones are not matched in weight,
prepare a balance tube(s) (if necessary) by pipetting a volume of water equal to that
of the sample into a previously used microconcentrator.
3. Place the microconcentrator opposite its balance tube in a 4 C microcentrifuge.
Microcentrifuge 20 to 60 min at the speed specied for the lter unit by the
manufacturer.
Centrifugation time is a function of the MWCO membrane used, i.e., lower cut-off re-
quires longer time.
A nonrefrigerated microcentrifuge may develop temperatures detrimental to protein sam-
ples when operated for extended periods; therefore it is usually best to have the micro-
centrifuge in a refrigerator or cold room for this operation, even though the ltration
rate is substantially reduced in the cold.
Microcon units can withstand forces of 13,000 g, whereas the Ultrafree units are
limited to 5,000 g. If the sample is not sufciently concentrated after 20 to 40 min,
centrifugation should be continued an additional 20 min to see if further volume reduction
occurs. Most centrifugal microconcentrators are designed to stop at a volume of 5 to
15 l. Typical centrifugation times to concentrate 500 l of 0.1% (w/v) BSA to 10 l at
4 C are 25 min for the Microcon-30 and 70 min for the Microcon-10. These times are
twice those observed at 25 C. Recovery is 95% in either instance. The microcentrifuge-
type concentrators usually are designed for use in xed-angle rotors, but some larger
concentrator units should be used only with swinging-bucket rotors. Devices like the
Ultrafree 4 and Ultrafree 15 units from Millipore can be run in either a xed-angle rotor
at 7,500 g or a swinging-bucket rotor at 4,000 g.
If desalting without concentration is required, remove the microconcentrator from the
microcentrifuge at the end of the centrifugation period, unscrew the sample reservoir
from the ltrate tube, then pour off the ltrate from the ltrate tube. Replace the reservoir
on the ltrate tube and add a sufcient amount of suitable buffer to restore the original
sample volume. Replace the cap and invert to mix. If the sample does not show the desired
nal ionic strength as determined by conductivity measurement, repeat the centrifugation
and readdition of buffer.

4. Once the desired degree of concentration has been achieved, remove the cap from
the unit and place the lter portion upside down into the retentate tube supplied by
the manufacturer.
5. Place the reversed unit back in the rotor and microcentrifuge 15 sec to drive the
concentrate down to the tip of the tube. Remove the unit from the microcentrifuge
and recover the sample from the retentate tube.
With some microconcentrators, adsorption of protein to the walls of the unit as well as
to the lter itself can be signicant when the sample is very dilute (25 g/ml). Passiva-
tion of the surfaces by soaking the entire unit overnight in 5% (v/v) Tween-20, followed
by thorough washing with distilled water, may help alleviate this problem. Suelter and
Deluca (1983) suggest that modication of the protein solvent by addition of glycerol or
Triton X-100 (below the critical micelle concentration) is superior to treatment of the
surfaces of the microconcentrator. A Millipore Protocol Note, No. PC1001EN00 (see In-
ternet Resources), provides data on the results of passivation of Microcon concentrators
following treatment with a variety of agents, including 5% (v/v) Tween-20 and 5% (v/v)
Triton X-100. While pretreatment with protein solutions such as 1% (w/v) BSA or 1% (w/v)
Extraction,
powdered milk are particularly effective, these can be recommended only when minute Stabilization,
amounts (1 g) of isotopically labeled proteins need to be concentrated or dialtered, and
and where trace amounts of these nonlabeled proteins would not be detrimental. Concentration

4.4.11
Current Protocols in Protein Science Supplement 38
 COMMENTARY
Background Information
Used primarily for removal of excess salts
or organic solvents (e.g., acetone or ethanol)
employed as precipitants in protein puri-
cation or analysis, molecular separations by
means of differentially permeable membranes
have been an integral part of protein biochem-
istry since the earliest purications. Mem-
branes of natural origin have long been sup-
planted for use in dialysis by regenerated cel-
lulose tubing. Relatively recent developments
in membrane technology have provided the
asymmetric designs now found in ultraltra-
tion membranes. This asymmetry refers to
the sidedness built into ultraltration mem-
branes, wherein a dense layer or skin with
a carefully controlled pore size is bonded to
one side of a thicker structural layer (com-
posed of the same or different material) that
has a large open-celled structure highly per-
meable to most molecules. A variety of ma-
terials have been used to fabricate these
semipermeable membranes, ranging from cel-
lulose and cellulose esters to polyethersul-
fone or polyvinylidene diuoride (PVDF). Be-
cause of proprietary interests, specic de-
tails concerning construction of these lters
particularly in regard to the more complex ma-
terials used in tangential-ow ultraltration
are not widely disseminated by their manu-
facturers. However, several companies (Mil-
lipore or their Amicon division, and Spec-
trum) make available extensive information
on their product lines and offer considerable
technical advice and literature pertaining to
their use. Much of this can be found on
their Web sites: http://www.millipore.com and
http://www.spectrumlabs.com.
All membranes, whether used for dialy-
sis or ultraltration, are characterized by their
molecular-weight cutoff (MWCO) value. This
is usually dened as the molecular weight of a
solute that is 90% prevented from penetrating
the membrane under a chosen set of condi-
tions. For some membranes, the MWCO value
is based on calibration using a mixture of dex-
trans, while for others it is based on calibration
with specic globular proteins. How readily a
particular protein is rejected by the membrane
is a function of the shape, hydration state, and
charge of the protein molecule. Hence, it is
Desalting, possible for two proteins of similar molecu-
Concentration, lar weight to have decidedly different reten-
and Buffer tion properties when a membrane with a stated
Exchange by
Dialysis and MWCO that is near their molecular weight is
Ultraltration used to separate them. For this reason, as well
4.4.12
Supplement 38
as uncertainties regarding applicability of cal-
ibration methods to the specic protein of in-
terest, it is recommended that MWCO values
be viewed as only close approximations. More-
over, MWCO values are not sharp; rather, there
is a gradual increase in retention as the size of
solute molecules approaches and exceeds the
average membrane pore size. Only at the point
where all pores are smaller than a particular
solute molecule is that molecule completely
excluded (Saltonstall, 1992a).
In standard dialysis procedures, the require-
ment is for a large amount of a diffusible
material to migrate through the membrane
while high-molecular-weight components are
retained. For simple dialysis, the MWCO cho-
sen is usually that which corresponds to half
(or less) the molecular weight for the smallest
protein to be retained. The rate of diffusion is
inuenced primarily by four factors: the con-
centration gradient of the diffusible material,
the diffusion constant of that material, the area
of the membrane, and the temperature. An in-
crease in any or all of these variables produces
more rapid dialysis; however the two that can
most readily be varied are the gradient and
the membrane area. The gradient should be
kept large by moderate stirring of the dialysis
buffer, which keeps the concentration of dif-
fusible solutes as low as possible at the mem-
brane exterior surface, as well as by frequent
changes of the buffer solution. It is also desir-
able to stir the contents of the dialysis bag
which speeds up internal diffusion and mini-
mizes concentration polarizationbut this is
not easily done without special equipment.
The primary reason that dialysis is commonly
used is that it is technically uncomplicated;
even though the technique is moderately slow,
arrangements intended to increase efciency
(e.g., rocking dialyzers) are rarely used (Mc-
Phie, 1971). Membrane area for a given length
of tubing is a direct function of the tubings
inated diameter (Table 4.4.1), such that a
1-cm-diameter bag will have one-half the sur-
face area of a 2-cm-diameter bag of the same
overall length. But since the volume of each
increases as the square of the radius, the vol-
ume that can be placed in the 2-cm-diameter
bag is four times that of the 1-cm bag. Thus if
only to maximize dialysis speed, it would be
better to place the sample that could be held in
a single 2-cm-diameter bag instead into four
1-cm-diameter bags since this would result in
an overall two-fold increase in surface area.
While this situation may not often be practical,
Current Protocols in Protein Science
it does illustrate the importance of using longer
but thinner tubing sections rather than shorter
but thicker sections when dialysis speed is
important.
The rate of movement for permeable solutes
in dialysis is difcult to predict, but the end
state that can be achieved is readily estimated.
Ignoring possible inequality between the con-
centrations of low-molecular-weight ions on
the two sides of a membrane that may result
from a Donnan effect, the equilibrium salt con-
centration is predictable from simple dilution
considerations. This is illustrated by the fol-
lowing example. If a 20-ml protein sample
in 2 M ammonium sulfate/50 mM potassium
phosphate is dialyzed against 1000 ml of a dial-
ysis buffer containing 50 mM potassium phos-
phate without ammonium sulfate, the dilution
factor will be 1020/20; therefore, at equilib-
rium, the (NH4 )2 SO4 concentration will be
0.039 M. If, after equilibration, this buffer is
replaced with 1000 ml fresh 50 mM potassium
phosphate, upon re-equilibration the ammo-
nium sulfate concentration will be <0.001 M.
If only 200 ml of 50 mM potassium phosphate
is used for each of the two buffer changes, how-
ever, the ammonium sulfate concentration in
the sample will approach 0.016 M. It is thus
highly desirable to change the dialysis buffer
several times or to use a large ratio of dialy-
sis buffer to sample. Both of these strategies
serve to maximize the concentration differen-
tial that drives the process of dialysis towards
the solute concentration present in the original
dialysis buffer.
Separation by means of dialysis or ultra-
ltration of two protein species that differ in
their molecular weights (and R values; see Crit-
ical Parameters) is possible under some condi-
tions, but generally will be incomplete unless
the size difference is large. Saltonstall (1992b)
has concluded that an efcient separation from
an initial 1:1 ratio to a nal 10:1 ratio would
require a ratio of retention values of 10:1 (e.g.,
0.9 versus 0.09), corresponding to a molecular-
weight ratio of 23:1 for several different types
of membrane.
Critical Parameters and
Troubleshooting
Membrane impurities
Dialysis membranes made from regener-
ated cellulose generally contain appreciable
amounts of heavy metals, with iron, zinc, and
lead present at levels in excess of 1 to 10 ppm.
They also contain 0.1% sulfur from the man-
ufacturing process and glycerol. Pretreatment
Current Protocols in Protein Science
may be necessary to remove these materials
when their presence is detrimental. Alterna-
tively, membranes that have been treated to re-
move the impurities can now be purchased, but
they must be kept wet. Cellulose ester mem-
branes do not contain these contaminants and
thus can be used directly; however they are less
tolerant of organic solvents and high pH than
are regenerated cellulose membranes.
Ultraltration
The advantage of desalting processes based
on ultraltration over those based on sim-
ple dialysis is that the rate of low-molecular-
weight solute removal is not determined by
a concentration differential, but rather by the
ow rate of solvent and the rejection of the so-
lute by the ultraltration membrane employed.
The rejection (R) of a solute is dened by the
relationship R = 1 (CF /CI ), where CF is the -
nal concentration of the solute (in the ltrate)
and CI is the initial concentration of the solute
(in the solution prior to ltration). In desalting
by dialtration, where the ltrate is replaced by
fresh dialysis buffer at the same rate at which
it is removed, a membrane is usually selected
whose MWCO is much larger than the molec-
ular weight of the solute to be removed. This
produces an R equal to zero for the permeable
solute, and, optimally, an R essentially equal to
one (i.e., total rejection) for the protein(s) of
interesta situation in which loss of protein
is minimized. When the dialtration involves
removal of a solute with an R equal to zero, the
degree of washout of the solute can be calcu-
lated from the volume of ltrate collected rel-
ative to the original sample volume according
to the equation: ln (CI /CR ) = (VF /VI ), where
CR is the nal concentration of the solute in
the retentate, VF is the volume of ultraltrate
collected (also equal to the volume of dialy-
sis buffer added back to the sample), and VI is
the initial sample volume (which remains con-
stant in dialtration because of readdition of
buffer). Using this equation, the volume of l-
trate that must be collected relative to sample
size (VF /VI ) can be estimated for any degree of
desalting required. Hence, to reduce the con-
centration of solute in the sample 10-fold (i.e.,
CI /CR = 10), the volume of ltrate collected
must be 2.3 the original sample volume: for
a 100-fold reduction, this volume ratio must
be 4.6.
Membranes for ultraltration are generally
selected on the basis of the MWCO needed Extraction,
to retain the protein of interest but allow the Stabilization,
maximum amount of other materials to pass and
Concentration
through. It is usually best to choose an MWCO
4.4.13
Supplement 38
 Table 4.4.2 Typical Flow Rates for Disk Type Ultraltration Membranesa

Membrane MWCO (kDa) Approximate ow rate at 25 C (ml/min/cm2 )

Water Protein solution

Amicon
YM3 3 0.07b 0.07c
YM10 10 0.2b 0.15c
YM30 30 0.9b 0.2c
PM10 10 2.5b 0.2c
PM30 30 4.0b 0.3c
Spectrum
Molecular/Por Type C 3 0.1d
d
Molecular/Por Type C 10 0.2
Millipore
Ultrafree 10 0.05e 0.05f
Ultrafree 30 0.4e 0.1f
a Addresses and telephone numbers of suppliers are provided in the SUPPLIERS APPENDIX.
b Determined at 55 psi.
c Determined at 55 psi using 0.1% albumin.
d Determined at 50 psi.
e Determined at 2000 g.
f Determined at 2000 g using 0.1% IgG.

value that is roughly one-half the molecular
weight of the smallest species to be retained.
This provides a reasonable margin of retention
whereby almost none of the protein of interest
should be lost, but at the same time provides the
largest difference between the MWCO value
and the molecular weight of the salts to be re-
moved, thereby maximizing ltration rate. Ta-
ble 4.4.2 lists ow rates for several types of
membrane disks. An additional factor to con-
sider, however, is the difference among mem-
branes in their chemical resistance. Where or-
ganic solvents are present in greater than trace
amounts, it may be necessary to avoid cellu-
lose acetate-type membranes in favor of regen-
erated cellulose types.
Concentration polarization
The term concentration polarization de-
scribes the concentration gradient of solutes
(mainly those to which the membrane is not
readily permeable) that forms immediately
above the membrane on the side under pres-
sure. This condition exists to some degree even
at low solute levels. Concentration polarization
Desalting, can be reduced by efcient remixing of the
Concentration,
and Buffer solution, either by stirring action (in stirred-
Exchange by cell systems) or tangential movement of so-
Dialysis and lution over the membrane (in tangential-ow
Ultraltration
systems). However, if the concentration of ma-
4.4.14
Supplement 38
terial within the gel layer at the membrane sur-
face reaches a value that is sufciently high, the
ltration rate becomes pressure-independent
and may fall to near zero.
One of the chief advantages of tangential-
ow ultraltration systems is that they al-
low better control of concentration polariza-
tion than stirred-cell systems. In tangential-
ow systems, concentration polarization is
controlled by varying the recirculation rate of
the retentate and adjusting the transmembrane
pressure. Flux of ltrate through the membrane
is a linear function of transmembrane pressure,
until gel formation on the surface becomes the
limiting factor. When this happens, increasing
the velocity across the membrane surface with-
out further increasing the transmembrane pres-
sure will improve ux.
Adsorption of protein to the membrane
In regard to the degree of nonspecic
adsorption of protein to membranes, some im-
portant differences exist among the various
materials used in membrane fabrication. Most
suppliers indicate that their cellulose mem-
branes exhibit the least nonspecic adsorp-
tion of protein; these are therefore recom-
mended for protein work, even though they
may have slower ltration rates than mem-
branes fabricated from other materials. The
Current Protocols in Protein Science
actual amount of protein bound depends on the
quantity present, its charge characteristics, and
the membrane area. Even with the best mem-
branes, losses of 1% to 5% are not uncommon
when dealing with total quantities of protein
in the range of 1 to 10 mg using a lter with a
43-mm diameter. The nature of the buffer can
also affect adsorption of protein. For example,
some membranes exhibit altered ow proper-
ties when high levels of ions are present. In
this regard, phosphate buffers seem to present
more of a problem than Tris buffers.
Precipitation of protein
Desalting sometimes results in precipita-
tion of a portion of the protein in the sam-
ple. Although the insoluble material can be re-
moved by centrifugation, it should not be dis-
carded until assays establish that the desired
protein has remained in solution. If the pre-
cipitated material includes the protein of inter-
est, it can often be redissolved in a solution of
higher ionic strength.
Anticipated Results
When carried out correctly, dialysis and di-
altration should produce a nearly complete
recovery of the protein of interest in a buffer
solution of the desired type and ionic strength.
In the case of dialysis, this requires that the
procedure be carried out for a sufcient time
period to equilibrate the salt concentrations in-
side and outside the dialysis bag. In the case
of dialtration with an asymmetric membrane,
this requires that a volume of ltrate be col-
lected that is equal to approximately ve times
the initial sample volume.
The degree of concentration to be achieved
by ultraltration should be that required for
subsequent work. However, it should be kept
in mind that once a protein concentration
>30 mg/ml has been reached, further concen-
tration is attained with increasing difculty and
production of very concentrated solutions can
therefore be slow. Recovery of sample follow-
ing concentration is generally >95%; failure
to achieve this value usually indicates leak-
age into the ltrate or nonspecic binding to
the membrane and/or concentration apparatus
itself.
Time Considerations
Dialysis through regenerated cellulose tub-
ing for desalting or buffer exchange usually
requires 8 to 12 hr for volumes 10 ml and
12 to 48 hr for volumes from 10 to 200 ml.
Dialysis time can be reduced by more frequent
buffer changes, by increasing the ratio of dial-
Current Protocols in Protein Science
ysis buffer to sample, by increasing the surface
area of the bag, and by monitoring the conduc-
tivity of the dialysis buffer to determine when
the desired ionic strength has been reached.
The rate of ultraltration, whether per-
formed for concentration or dialtration,
varies considerably as a function of the
MWCO and surface area of the membrane and
the initial sample concentration. A 10-fold re-
duction in sample volume (or 10-fold reduc-
tion in the concentration of a low-molecular-
weight impurity by dialtration) can usually
be accomplished in 1 to 4 hr.
Literature Cited
McPhie, P. 1971. Dialysis. Methods Enzymol.
22:23-32.
Saltonstall, C.W. 1992a. Separations with dialysis
and ultraltration: Theoretical and practical con-
siderations. Am. Biotechnol. Lab. 10:32-34.
Saltonstall, C.W. 1992b. Dialysis and ultraltra-
tion: Estimating quantitative separations. Am.
Biotechnol. Lab. 10:18-20.
Suelter, C.H. and Deluca, M. 1983. How to pre-
vent losses of protein by adsorption to glass and
plastic. Anal. Biochem. 135:112-119.
Key References
Pohl, T. 1990. Concentration of proteins and re-
moval of solutes. Methods Enzymol. 182:68-83.
A thorough review of the topics covered here.
Scopes, R.K. 1994. Protein Purication: Principles
and Practice, 3rd ed. pp. 14-21. Springer-Verlag,
New York.
Covers concentration and dialysis in the context of
protein purication techniques.
Schratter, P. 1996. Purication and concentration by
ultraltration. Methods Mol. Biol. 59:115-134.
Additional description of protein concentration
methodology.
Doonan, S. 1996. Concentration of extracts. Meth-
ods Mol. Biol 59:95-102.
Additional description of protein concentration
methodology.
Internet Resources
http://www.millipore.com/publications.nsf/
dda0cb48c91c0fb6852567430063b5d6/
21faf0f81edcbb68852568f00053d6a2/$FILE/
ATTDMXFA/PC1001EN00.pdf
Millipore Protocol Note, No. PC1001EN00, regard-
ing results from passivation of Microcon concentra-
tors following treatment with a variety of agents.
Contributed by Allen T. Phillips and
Mark W. Signs
Extraction,
Pennsylvania State University
Stabilization,
University Park, Pennsylvania and
Concentration
4.4.15
Supplement 38