Acrylamide Polymerization — A Practical Approach
Paul Menter, Bio-Rad Laboratories, 2000 Alfred Nobel Drive,
Hercules, CA 94547 USA
Introduction
The unparalleled resolution and flexibility possible with polyacrylamide
gel electrophoresis (PAGE) has led to its widespread use for the separation
of proteins and nucleic acids. Gel porosity can be varied over a wide
range to meet specific separation requirements. Electrophoresis gels and
buffers can be chosen to provide separation on the basis of charge, size,
or a combination of charge and size.
The key to mastering this powerful technique lies in the
polymerization process itself. By understanding the important
parameters, and following a few simple guidelines, the novice
can become proficient and the experienced user can optimize
separations even further.
This bulletin takes a practical approach to the preparation of polyacrylamide
gels. Its purpose is to provide the information required to achieve reproducible,
controllable polymerization. For those users interested only in the “bare
essentials,” the Polymerization Protocols can be used as a laboratory
guide.
Mechanism of Polymerization
Polyacrylamide gels are formed by copolymerization of acrylamide and bis-acrylamide
(“bis,” N,N'-methylene-bisacrylamide). The reaction is a vinyl
addition polymerization initiated by a free radical-generating system
(Chrambach 1985). Polymerization is initiated by ammonium persulfate and
TEMED (tetramethylethylenediamine): TEMED accelerates the rate of formation
of free radicals from persulfate and these in turn catalyze polymerization.
The persulfate free radicals convert acrylamide monomers to free radicals
which react with unactivated monomers to begin the polymerization chain
reaction (Shi and Jackowski 1998). The elongating polymer chains are randomly
crosslinked by bis, resulting in a gel with a characteristic porosity
which depends on the polymerization conditions and monomer concentrations.
Riboflavin (or riboflavin-5'-phosphate) may also be used as a source
of free radicals, often in combination with TEMED and ammonium persulfate.
In the presence of light and oxygen, riboflavin is converted to its leuco
form, which is active in initiating polymerization. This is usually referred
to as photochemical polymerization.
Polyacrylamide Gel Polymerization
Purity of Gel-Forming Reagents
Acrylamide
Gel-forming reagents include the monomers, acrylamide and bis, as well
as the initiators, usually ammonium persulfate and TEMED or, occasionally,
riboflavin and TEMED. On a molar basis, acrylamide is by far the most
abundant component in the monomer solution. As a result, acrylamide may
be the primary source of interfering contaminants (Dirksen and Chrambach
1972). Poor-quality acrylamide contains significant amounts of the following
contaminants:
1. Acrylic acid — Acrylic acid is the deamidation product of
acrylamide. Acrylic acid will copolymerize with acrylamide
and bis, thereby conferring ion exchange properties on the
resulting gel. This can lead to local pH changes in the gel and
cause artifacts such as aberrant relative mobility, precipitation of
some proteins and nucleic acids, streaking or smearing of
bands, and run-to-run irreproducibility. In acrylamide, acrylic acid
should be below 0.001% (w/w). This is determined by direct
titration, and supported by both conductivity and
pH measurement.
2. Linear polyacrylamide — Contaminants with catalytic properties
may cause what appears to be autopolymerization during the
production, processing, or storage of marginally pure acrylamide.
This results in the presence of linear polyacrylamide in the dry
monomer. Linear polyacrylamide will affect polymerization, since it
serves as a nucleus for polymerization. The most important effect
is the loss of reproducibility in gel porosity and relative mobilities
of proteins and nucleic acids. Linear polyacrylamide is detected
as water or alcohol insolubles and should be <0.005% (w/w).
3. Ionic contaminants — Ionic contaminants can include both
inhibitors and accelerators of polymerization. Aside from
acrylic acid, the most notable ionic contaminants are metals
such as copper, which can inhibit gel polymerization.
Metals can also poison enzymes, alter the relative mobility
of metal binding proteins such as calmodulin, and inhibit
digestion of electrophoretically purified nucleic acids by
restriction and modification enzymes. Ionic contaminants
are detected indirectly by their effects on chemical and
photochemical polymerization, and by the conductivity of
monomer solutions.
bis-Acrylamide
Bis is present in much smaller quantities than acrylamide in monomer solutions.
However, improperly purified bis contains some of the same contaminants
as acrylamide. These include products of autopolymerization and ionic
contaminants, which have the same deleterious effects, and can be detected
in the same ways, as the corresponding acrylamide contaminants.
Initiators
Chemical polymerization is initiated by ammonium persulfate, while photochemical
polymerization is initiated by riboflavin (or riboflavin-5'-phosphate),
or by a combination of riboflavin and ammonium persulfate. Initiation
and polymerization are catalyzed by TEMED. Because polymerization is initiated
by the generation of free radicals from persulfate or riboflavin, it is
not surprising that these compounds are reactive, and prone to oxidation
or decomposition. The contaminants of the initiators tend to be the products
of their own breakdown as well as other contaminating compounds.
TEMED is subject to oxidation, which causes the gradual
loss of catalytic activity. This process is greatly accelerated
by contaminating oxidizing agents. TEMED that contains
oxidation products is characterized by a yellow color. The
practical consequences of the oxidative process are the
requirement for greater amounts of TEMED to achieve
adequate polymerization, and a gradual loss of TEMED
reactivity with time. TEMED is also very hygroscopic and will
gradually accumulate water, which will accelerate oxidative
decomposition. TEMED with maximum activity and shelf life
is obtained by redistillation immediately prior to bottling,
resulting in a product that is clear, water free, and greater
than 99% pure (14.4 M).
Ammonium persulfate is also very hygroscopic. This property
is particularly important, since ammonium persulfate begins
to break down almost immediately when dissolved in water.
Therefore, the accumulation of water in ammonium persulfate
results in a rapid loss of reactivity. This is why ammonium
persulfate solutions should be prepared fresh daily. Persulfate
is consumed in the polymerization reaction. Excess persulfate
can cause oxidation of proteins and nucleic acids. This
oxidation problem can be avoided if inhibitor-free gel-forming
reagents are used, and ammonium persulfate is used at the
recommended levels.
Contaminants in Buffers
Contaminants in buffer reagents (Tris, borate, acetate, glycine, etc.),
gel additives (SDS, urea, etc.), and laboratory water can have a profound
effect on polymerization. The most common contaminants of these reagents
are metals, non-buffer ions, and breakdown products. The most frequent
effect of these contaminants is to inhibit polymerization. When polymerization
is partially inhibited, the resulting gel will have greater porosity than
intended, and molecules will have greater mobilities. Furthermore, control
over polymerization reproducibility is compromised.
Initiator Type and Concentration
Initiators are the effectors of polymerization. Of course, the rate of
polymerization depends on the concentration of initiators, but more importantly,
the properties of the resulting gel also depend on the concentration of
initiators. Increasing the concentration of initiators (e.g., ammonium
persulfate and TEMED) results in a decrease in the average polymer chain
length, an increase in gel turbidity, and a decrease in gel elasticity.
In extreme cases, excess initiator can produce a gel solution that does
not appear to polymerize at all. This is due to the formation of polymer
chains so short that visible gelation does not take place and the polymer
stays in solution. The only indication that a reaction has taken place
is an increase in viscosity.
Excess ammonium persulfate and TEMED have other
effects, including oxidation of sample proteins (especially
sulfhydryl-containing compounds) and changes in buffer pH.
Excess TEMED can increase buffer pH, react with proteins
(Dirksen and Chrambach 1972; Chrambach et al. 1976),
and alter the banding pattern (Gelfi and Righetti 1981a).
Ammonium persulfate acts as a buffer between pH 8 and 9.
Potassium persulfate is recommended instead of ammonium
persulfate in weakly buffered basic systems (~pH 9).
Excess riboflavin may cause the oxidation of some
compounds, especially sulfhydryl-containing compounds
(Dirksen and Chrambach 1972), and can denature proteins
(Righetti et al. 1981).
Reducing the concentration of initiators results in longer
polymer chain lengths, lower turbidity, and greater elasticity.
These are desirable properties. However, lower initiator
concentrations also mean slower polymerization. If polymerization
is too slow, oxygen will begin to enter the monomer
solution and inhibit polymerization, resulting in gels which are
too porous and mechanically weak. Inhibition will be especially
pronounced at surfaces exposed to air, or at the surfaces
of combs and spacers, which appear to trap air at their
surfaces. The remaining unpolymerized monomer can react
with alpha amino, sulfhydryl, and phenolic hydroxyl groups of
proteins (Allison et al. 1974; Chrambach et al. 1976; Dirksen
and Chrambach 1972).
For discontinuous systems which employ a stacking gel (e.g.,
Laemmli system), optimal polymerization of the overlaid lower
gel (resolving gel) is achieved when visible gelation takes
place 15–20 min after the addition of the initiators ammonium
persulfate and TEMED (note that polymerization continues
long after visible gelation takes place; see Figure 1). For
stacking gels and continuous systems (which do not contain
stacking gels) — i.e., any gel which is not overlaid — optimal
polymerization results when visible gelation takes place
in 8–10 min. Higher initiator concentrations and faster
polymerization are required in these cases because of the
inhibitory effect of atmospheric oxygen associated with the
comb. In any case, conversion of monomer to polymer
should be greater than 95%. If gelation takes longer than
20 min, the inhibitory effects of atmospheric oxygen will
begin to appear.
As a general rule, use the lowest catalyst concentrations that
will allow polymerization in the optimal period of time. In the
case of ammonium persulfate/TEMED-catalyzed reactions,
for example, approximately equimolar concentrations of both
catalysts in the range of 1 to 10 mM are recommended.
Riboflavin is often used as an initiator along with TEMED, or
with TEMED and ammonium persulfate. The major advantage
of riboflavin is that it is active in very low concentrations
(~5–10 µg/ml). Thus, when riboflavin is used with TEMED and
ammonium persulfate, the total amount of initiator required
(sum of the three initiators) is less. Given the possible effects
of initiators on buffer pH, riboflavin-based initiator systems are
useful for poorly buffered systems such as electrofocusing
gels, in which the only buffering components are ampholytes.
Visible gelation takes longer in riboflavin-based initiator
systems, usually 30–60 min. Oxygen does not have the
dramatic inhibitory effect on riboflavin-based initiator systems
that it has on TEMED/ammonium persulfate systems. This is
presumably due to the oxygen-scavenging property of
riboflavin. As a result, longer gelation time can be tolerated.
In chemical polymerization, visible gelation occurs in 15–20 min
and polymerization is essentially complete in 90 min. In photochemical
polymerization, however, visible gelation takes 30–60 min and complete
polymerization requires up to 8 hr (Righetti et al. 1981). Shorter times
lead to more porous and elastic gels, increased risk of protein modification,
and pore size irreproducibility.
Temperature
Temperature control is critical for reproducibility of acrylamide polymerization.
Temperature has a direct effect on the rate of gel polymerization; the
polymerization reaction is also exothermic. Consequently, the generated
heat drives the reaction more quickly. Thus, gelation usually occurs very
rapidly once polymerization begins.
Temperature also affects the properties of the gel (Chen and
Chrambach 1979). For example, polymerization at 0–4°C
results in turbid, porous, inelastic gels, and reproducibility is
difficult to achieve. These properties may be due to increased
hydrogen bonding of monomer at low temperatures. Gels
polymerized at 25°C are more transparent, less porous, and
more elastic. However, if the polymerization temperature is
too high, short polymer chains are formed and the gels are
inelastic. This is thought to be due to increased polymer
chain termination at higher temperatures.
A temperature of 23–25°C is optimal (as well as most
convenient) for polymerization. It is important that the monomer
solution and the gel mold (e.g., glass plates or tubes) be at
the optimal temperature when the gel is poured. Furthermore,
reproducibility is dependent on using the same temperature
each time gels are poured.
Since monomer solutions are usually stored at 4°C along with buffer
concentrates, it is important to allow the monomer gel solution, once
prepared, to equilibrate to room temperature before being evacuated (if
cold solutions are placed under vacuum they tend to stay cold).
Oxygen
The formation of polyacrylamide gels proceeds via free radical polymerization.
The reaction is therefore inhibited by any element or compound that serves
as a free radical trap (Chrambach 1985). Oxygen is such an inhibitor.
Oxygen, present in the air, dissolved in gel solutions, or adsorbed to
the surfaces of plastic, rubber, etc., will inhibit, and in extreme cases
prevent, acrylamide polymerization. Proper degassing is critical for reproducibility.
Therefore, one of the most important steps in the preparation of polyacrylamide
gels is the evacuation, or “degassing” of gel solutions immediately
prior to pouring the gel. This is done by placing the flask of gel solution
in a vacuum chamber or under a strong aspirator. In some cases, a vacuum
pump may be required.
Buffer stock solutions and monomer stock solutions are usually
stored at 4°C. Cold solutions have a greater capacity for
dissolved oxygen. The process of degassing is faster and
more complete if the gel solution is brought to room
temperature (23–25°C)‚ before degassing begins.
Furthermore, if a cold gel solution is placed under vacuum,
the process of evacuation tends to keep the solution cold.
Pouring a gel with a cold solution will have a substantial
negative effect on the rate of polymerization and on the
quality of the resulting gel.
Polymerization in which riboflavin is used as one of the
initiators calls for degassing. The conversion of riboflavin from
the flavo to the leuco form (the species active in initiation)
actually requires a small amount of oxygen (Gordon 1973).
This explains why polymerization initiated primarily by riboflavin
can be completely blocked by exhaustive degassing. However,
oxygen in excess of that needed to convert riboflavin to the
active form will inhibit polymer chain elongation, as it does in
reactions initiated only by ammonium persulfate and TEMED.
Thus, while degassing is still important for limiting inhibition,
it must not be so extensive that it prevents conversion of
riboflavin to the active form. For polymerization initiated by
riboflavin/TEMED, or riboflavin/TEMED/ammonium persulfate
systems, degassing should not exceed 5 min.
A consequence of the interaction of riboflavin with oxygen is
that riboflavin seems to act as an oxygen scavenger. This is
supported by the observation that the addition of riboflavin
(5 µg/ml) to stacking gel solutions containing ammonium
persulfate/TEMED initiators results in cleaner, more uniform
polymerization at gel surfaces exposed to oxygen (such as
combs). The same effect could likely be achieved by more
thorough degassing of solutions without riboflavin.
Whether using chemical polymerization (ammonium persulfate/TEMED) or
photochemical polymerization (riboflavin/TEMED or riboflavin/TEMED/ammonium
persulfate initiators), reproducible gel quality and separation characteristics
require careful attention to gel solution temperature before degassing,
and to degassing time, temperature, and vacuum. These parameters should
be kept constant every time gels are prepared.
pH
The majority of electrophoresis systems are buffered at neutral or basic
pH, at which the common initiators, ammonium persulfate, TEMED, and riboflavin,
are effective. Riboflavin is the better choice for polymerization at low
pH (Shi and Jackowski 1998); however, at low pH, TEMED may become protonated.
This can result in slower polymerization, since the free base form of
TEMED is required for initiation. For acidic buffer systems, alternative
initiator systems are sometimes used (Andrews 1990).
Alternative Crosslinkers
PDA (piperazine di-acrylamide), a crosslinking agent that can be substituted
for bis in polyacrylamide gels, offers several advantages for electrophoresis.
These include reduced background for silver staining, increased gel strength,
and higher-resolution gels. PDA can be substituted for bis on a weight
basis without changing polymerization protocols.
Crosslinkers other than bis and PDA may be used for specialized purposes,
the most common of which is gel solubilization during post-electrophoresis
recovery of proteins or nucleic acids. These crosslinkers include DATD
(diallyltartardiamide), DHEBA (dihydroxyethylene-bis-acrylamide), and
BAC (bis-acrylylcystamine). Alternative crosslinkers may be more or less
reactive in polymerization than bis. Therefore, some adjustment in the
concentration of initiators may be necessary to achieve optimal polymerization.
For a discussion of alternative crosslinkers, see Gelfi and Righetti (1981b).
Gel Additives
The most common gel additives include SDS (sodium dodecyl sulfate), Triton*
X-100 detergent, and chaotropic agents such as urea and formamide. Detergents
can be added to most common buffer systems without significantly affecting
polymerization. Agents such as urea and formamide, however, cause the
formation of smaller pore-size gels than would be formed in their absence
(urea is often a component of gel systems used to separate small proteins
and peptides). This may be due to the disruption of hydrogen bonds between
monomer molecules during polymerization. Smaller pore size may also be
achieved at higher polymerization temperatures, an effect also attributed
to hydrogen bond disruption. Contaminants of gel additives can affect
polymerization. Nonionic additives such as urea, formamide, and Triton
X-100 can be deionized with a mixed-bed ion exchange resin. Use 10 gm
Bio-Rad Ag 5O1 X-8 resin per 100 ml additive solution and let sit overnight.
However, removal of nonionic contaminants from nonionic reagents is not
practical. Therefore, all additives should be qualityassured for electrophoresis.
Time
Although visible gelation occurs in 15–20 min for chemical polymerization
and 30–60 min for photochemical polymerization, polymerization continues
much longer (see Figure 1). Ammonium persulfate/TEMED-initiated reactions
should be allowed to proceed for 2 hr to ensure maximum reproducibility
in gel pore size. Photochemical polymerization (riboflavin-based initiator
system) usually proceeds more slowly than chemical polymerization, and
is also dependent on light intensity (Shi and Jackowski 1998). However,
riboflavin is usually used for polymerization of electrofocusing gels
in which separation is based on charge, and for which gel porosity is
of secondary importance. Thus these gels can be used shortly after visible
gelation without being affected by slight variations in porosity.
Monomer Concentration
The practical range for monomer concentration is between 3%T and 30%T,
where %T refers to % (w/v) of total monomer (acrylamide + bis) in solution.
A higher concentration of monomer results in faster polymerization. Therefore,
changing from 5% gels to 30% gels will probably allow a reduction of 20–50%
in the concentration of initiators.
Polymerization Protocols
There are 2 major initiator formulations for acrylamide polymerization.
The first, for chemical polymerization, is used for SDS-PAGE and DNA sequencing.
Chemical polymerization employs ammonium persulfate and TEMED as initiators.
The second, for photochemical polymerization, is used primarily for horizontal
electrofocusing gels. Photochemical polymerization calls for riboflavin
as well as ammonium persulfate and TEMED. Riboflavin phosphate can be
substituted for riboflavin. Riboflavin phosphate is often preferred for
its greater solubility.
Preparation for Polymerization
1. Prepare 10% ammonium persulfate shortly prior to use (prepare fresh
daily). TEMED is used undiluted. Prepare 0.1% riboflavin (or riboflavin
phosphate, which is more soluble) if photopolymerization will be performed.
2. Combine buffer stock solution, monomer stock solution, and water in
the appropriate proportions in an Erlenmeyer flask. Since stock solutions
are usually stored at 4°C, the gel solution should be allowed to warm
to room temperature before degassing.
3. Prepare the gel casting mold, i.e., plates, spacers, and clamps for
gel casting. Be sure they are neither hot nor cold.
4. Once the gel solution is prepared and brought to room temperature (23–25°C),
degas the solution under a vacuum of 125 torr or better for 15 min at
room temperature (for systems in which constant agitation is used during
degassing, 10 min is sufficient). Longer periods of degassing are generally
not deleterious, although long degassing will result in somewhat faster
polymerization.
Chemical Polymerization in Discontinuous Systems — Lower (Resolving)
Gel
In a discontinuous system, such as that of Laemmli, the resolving gel
is polymerized first. Then, the stacking gel is cast on top of the resolving
gel. Use the following protocol to prepare resolving gels for all discontinuous
systems (see the next section for preparation of stacking gels).
Swirl the solution gently but thoroughly. Holding the flask by
the neck with one hand, swirl it 8 to 10 cycles. This mixes
the initiators completely without introducing too much oxygen.
Swirling too little can result in uneven polymerization.
Cast the gel by introducing the monomer solution into the gel
mold in a steady stream to minimize the introduction of oxygen.
Overlay the monomer solution using water, isoamyl alcohol,
or water-saturated isobutyl alcohol to exclude oxygen from
the surface.
Allow polymerization to occur at room temperature at least 90 min prior
to use (see Figure 1).
Chemical Polymerization in Continuous Systems and Stacking Gels
Continuous systems consist of a single gel. Continuous systems are used
for some types of protein electrophoresis, and for DNA sequencing. Stacking
gels are part of discontinuous systems. These gels have in common contact
with the well-forming comb and greater exposure to molecular oxygen at
the surface. Use the following levels of initiators for continuous systems
and stacking gels.
Swirl the solution gently but thoroughly.
Cast the gel and insert the well-forming comb without trapping
air under the teeth.
Allow polymerization to occur at room temperature at least 90 min prior
to use (see Figure 1).
Photochemical Polymerization
This protocol is recommended for isoelectric focusing (IEF) gels. Since
molecules remain in the IEF gel during electrophoresis, excess ions from
initiators can cause distortion of bands. Photochemical initiation is
recommended for IEF gels because it is effective at low initiator concentrations.
1. When the gel solution is prepared and brought to room
temperature (23–25°C), degas the solution under a vacuum of
125 torr or better for 2 min at room temperature (for systems in
which constant agitation is used for degassing, 1 min
is sufficient).
2. Add initiators as follows:
3. Swirl the solution gently but thoroughly.
4. Cast the gel and allow polymerization to occur for at least
2 hr for isoelectric focusing gels. If separation is to be based
on size, allow photochemically initiated gels, to polymerize
for 8 hr under light from a nearby fluorescent lamp, (Righetti
et al. 1981).
Polymerization Analysis
There are several ways to assess the extent and reproducibility of polymerization.
One of the easiest methods is to routinely monitor the time required for
visible gelation. There are several factors which affect the polymerization
rate. A significant change in the time required for visible gelation indicates
that one of the parameters has changed.
The polymerized gel should be inspected for evidence of inhibition or
nonuniform polymerization. A swirled or “schlieren” pattern,
for example, indicates that polymerization was too fast or that the polymerization
initiators were not mixed thoroughly with the monomer solution prior to
casting the gel.
Polymerization Profile
As acrylamide polymerizes, UV-absorbing double bonds are eliminated. The
progress of a reaction can therefore be followed by monitoring absorbance
at 260 nm. As the reaction proceeds, the UV absorbance drops. Absorbance
increases with the amount of unreacted monomer.
Figure 1 shows a UV profile of chemical polymerization for
2 samples of acrylamide polymerized under identical conditions
in a quartz cuvette. Sample A was an “enzyme grade”
acrylamide with a conductivity (50% w/w) of 3.75 µS. Sample B
was Bio-Rad’s electrophoresis-purity acrylamide with a
conductivity of 0.56 µS. As the figure shows, polymerization is
largely complete after about 90 min, even though the
reaction proceeds to a small extend beyond that time.
While sample A began to polymerize faster, sample B
polymerized more completely, as indicated by the lower
final UV absorbance.
Contaminants in acrylamide may be accelerators or inhibitors of
polymerization. Therefore, initiation of polymerization, as
indicated by reduced absorbance, may be faster with crude
acrylamide than with highly refined acrylamide. However, the
most important consideration is the completeness of
polymerization. Polymerization of highly refined acrylamide
may be initiated more slowly, but conversion of monomer to
polymer, as indicated by the low final absorbance, is more
complete. Therefore, less residual monomer remains. Complete
polymerization is critical for reproducibility in gel porosity.
Gel Exclusion Limit Determination
Estimation of protein molecular weight by SDS-PAGE is a widely employed
procedure. The relative mobility of a protein in an SDS-PAGE gel is related
to its molecular weight. A standard curve is constructed with proteins
of known molecular weight by plotting the logarithms of their molecular
weights versus the relative mobilities of the proteins. The relative mobility
of a protein of unknown molecular weight is then fitted to the curve to
determine its molecular weight.
A standard curve can be extrapolated to give the y-intercept,
which represents the molecular weight exclusion limit of that
particular gel. That is, proteins with a molecular weight greater
than the y-intercept value will show zero mobility and will be
excluded from the gel matrix.
Poorly polymerized gels have greater porosity due to incomplete
chain elongation and crosslinking. As a result, the exclusion limit
will be greater than for a well-polymerized gel of the same
percent acrylamide. Furthermore, when polymerization is
incomplete, exclusion limits are irreproducible. Use of highly
purified gel-forming reagents and proper polymerization
technique will result in the lowest and most reproducible
exclusion limits for a given percent total monomer.
Figure 2 shows a typical curve obtained by plotting log
molecular weight versus relative mobility following SDS-PAGE
for a group of standard proteins. The antilog of the y-intercept
value of this plot is 115,000 as determined by linear regression
analysis. The approximate molecular weight exclusion limit of
the gel is thus 115,000. The y-intercept value should be
considered approximate because it depends upon the
relative mobility of the proteins used as standards.
Although the y-intercept value will be different for every gel
acrylamide percentage, and slightly different for every set of
standards, the value should be highly reproducible from gel to
gel if the same acrylamide percentage and standards are used.
Thus, monitoring the y-intercept of the log molecular weight vs.
relative mobility plot is an excellent assessment of reproducibility
in polymerization technique.
* %C = (grams crosslinker x 100)/(grams monomer + grams crosslinker)
Handling of Acrylamide
Use good laboratory practices, work in a well ventilated area and wear
proper personnel proctective equipment. Refer to the MSDS for further
information.
Reagent Storage and Shelf Life
Acrylamide and bis-acrylamide — Electrophoresis-purity acrylamide
and bis can be stored dry at room temperature (23–25°C) for
at least 1 year.
Ammonium persulfate and potassium persulfate — These
initiators can be stored tightly sealed at room temperature for
at least 1 year. Solutions should be made fresh daily, since
persulfate in solution decomposes rapidly. Persulfate is a
strong oxidizing agent. Disposal should be in accordance
with local regulations.
TEMED — This initiator can be stored tightly closed either at
4°C or at room temperature for at least 6 months.
After 10 to 12 months, a significant reduction in reactivity
requires an increase in the concentration required for proper
polymerization. This loss of reactivity is probably due, at least
in part, to the gradual accumulation of water.
Riboflavin and riboflavin-5'-phosphate — These
photoinitiators can be stored dry at room temperature for at
least 1 year. In aqueous solution, they are stable for at least
1 month if kept in the dark at 4°C. Riboflavin phosphate is
usually preferred because of its greater solubility.
References
Allison JH et al., Effect of N,N,N’,N’-tetramethylethylenediamine
on the migration of proteins in SDS polyacrylamide gels, Anal Biochem
58, 592–601 (1974)
Andrews AT, Acid-urea detergent gels, pp 141–143 in Electrophoresis
Theory,
Techniques, and Biochemical and Clinical Applications, 2nd ed, Oxford
Science
Publications, Oxford (1990)
Chen B and Chrambach A, Estimation of polymerization efficiency in the
formation of polyacrylamide gel, using continuous optical scanning during
polymerization, J Biochem Biophys Methods 1, 105–116 (1979)
Chrambach A et al., Analytical and preparative polyacrylamide gel
electrophoresis. An objectively defined fractionation route, apparatus,
and procedures, Methods Protein Sep 2, 27–144 (1976)
Chrambach A, The Practice of Quantitative Gel Electrophoresis, VCH,
Deerfield Beach (1985)
Dirksen ML and Chrambach A, Studies on the redox state in poly acrylamide
gels, Sep Sci 7, 747–772 (1972)
Gelfi C and Righetti PG, Polymerization kinetics of polyacrylamide gels
I. Effect
of different cross-linkers, Electrophoresis 2, 213–219 (1981a)
Gelfi C and Righetti PG, Polymerization kinetics of polyacrylamide gels
II. Effect
of temperature, Electrophoresis 2, 220–228 (1981b)
Gordon AH, Electrophoresis of Proteins in Polyacrylamide and Starch Gels,
2nd
ed, Elsevier/North-Holland Biomedical Press, Amsterdam (1975)
Righetti PG et al., Polymerization kinetics of polyacrylamide gels. III.
Effect of
catalysts, Electrophoresis 2, 291–295 (1981)
Shi Q and Jackowski G, One-dimensional polyacrylamide gel electrophoresis,
pp 1–52 in Hames BD (ed) Gel Electrophoresis of Proteins: A Practical
Approach, 3rd edn, Oxford University Press, Oxford (1998)
* Triton is a trademark of Union Carbide Chemicals and Plastics Technology
Corp.
back to top