acrylamide polymerization
tfitzwater at nexstar.com
tfitzwater at nexstar.com
Mon Sep 22 11:33:17 EST 1997
(subscript: The best source for information about acrylamide
polymerization
is BioRad Bulletin 1156: "Polyacrylamide gels are formed
by
co-polymerization of acrylamide and bis-acrylamide. The reaction is
a
vinyl addition polymerization initiated by a free
radical-generating
system. Polymerization is initiated by TEMED
(tetramethylethylenediamine)
and APS (ammonium persulfate). The TEMED acts as an electron carrier
to
activate the acrylamide monomer, providing an unpaired electron to
convert
the acrylamide monomer to a free radical. The activated monomer
then
reacts with unactivated monomer to begin the polymer chain reaction.
The
elongating polymer chains are randomly crosslinked by bis, resulting
in
closed loops and a complex web polymer with a characteristic porosity
which
depends on the polymerization conditions and monomer concentration." )
(subscript: Gels may not completely polymerize, depending on factors
such
as concentration of initiator, temperature, pH, additives,
breakdown
products and impurities in the chemicals and water used. Light and
high
temperature can cause autopolymerization of linear acrylamide which
affects
the reproducibility of gel formation.)
(subscript: )
(subscript: Acrylamide purity)
(subscript: Poor quality acrylamide contains acrylic acid (a
deamidation
product) which will copolymerize and cause local pH changes in the
gel
leading to streaking and smearing of bands. Linear
polyacrylamide
contaminants effect reproducibility. Ionic contaminants can include
both
inhibitors and accelerators of polymerization.)
(subscript: During storage at room temperature, especially in
water,
acrylamide breaks down into acrylic acid, polyacrylamide, )(subscript:
BBB)
(subscript: -nitrolotrispropionamide and ammonia. The pH of 10%
acrylamide
dissolved in 0.1 N NaCl should be 6.50 +/- 0.5 The pH of
1%
N,N'-Methylene-Bis-Acrylamide dissolved in Type I water should be
)
(subscript: >)(subscript: 5.5.)
(subscript: Urea)
(subscript: Urea (NH2)2CO has a formula weight of 60.06. The problem
with
the use of urea in protein gels is the accumulation of cyanate ions
in
stock solutions due to chemical isomerization. The cyanate reacts
with
amino groups to form stable carbamylated derivatives which modify
the
charge of proteins. The free amino groups of Tris neutralize the
cyanate
ions. Where possible, the heating of urea-containing solutions should
be
avoided as cyanate ions formation is accelerated with
increasing
temperature. Urea slowly decomposes in water, forming ammonia and
very
reactive cyanate ions. Urea solutions may be pretreated with
mixed-bed
resins or mixed with small amounts of Tris that reacts with the
cyanate
ions. )
(subscript: Tris)
(subscript: It is essential that Tris buffers are prepared with Tris
base.
After Tris base has been dissolved in Type I water, the pH of the
solution
should be adjusted with HCl. If Tris-HCl or Trizma are used to
prepare
buffers, the concentration of salt will be too high and material
will
migrate anomalously, yielding extremely diffuse bands. )
(subscript: Tris-base is oxidized during electrophoresis to a
UV-absorbing
compound.)
(subscript: )
(subscript: APS (ammonium persulfate))(subscript: MW 228.22)
(subscript: APS is a strong oxidant. It is an irritant by inhalation
or
contact with skin or eyes. )
(subscript: APS (ammonium persulfate) is very hygroscopic and reactive
by
nature and is prone to oxidation and decomposition. It begins to
break
down almost immediately when dissolved in water. Optimally, APS should
be
prepared fresh daily. Certainly, 10% stocks should be replaced every
month
for minimal reproducibility. Listen for a crackling sound when
adding
water to dry APS. Material that has broken down due to high humidity
or
liquid contamination will no longer crackle when moistened. Persulfate
is
consumed in the polymerization since it provides the free radicals
that
drive polymerization, but excess APS can cause oxidation of proteins
and
nucleic acids. Prerunning gels keeps excess APS traveling ahead of
the
sample. BioRad recommends 50 ?l of fresh 10% APS per 10 ml of gel
solution
for a final concentration of 0.05%. I use 100 ?l of fresh 10% APS per
10
ml of gel solution for a final concentration of 0.1%. )
(subscript: TEMED)(subscript: (tetramethylethylenediamine))
(subscript: TEMED is neurotoxic and extremely flammable. TEMED
is
extremely destructive to mucous membranes and upper respiratory tract,
eyes
and skin. Inhalation may be fatal. Prolonged contact can cause
severe
irritation or burns.)
(subscript: Use of a fume hood is recommended.)
(subscript: TEMED (tetramethylethylenediamine) is subject to
oxidation
which causes a gradual loss in catalytic activity. It is hygroscopic
and
accumulates water which accelerates oxidative decomposition. It should
be
discarded as chemical waste when oxidation products begin to give it
a
yellow color. For this reason, it is recommended that clear tips be
used
to add TEMED.)
(subscript: TEMED also effects the pH gradient of IEF gels. It
introduces
fixed charges into the gel matrix which cause gradient drift towards
the
anode. Non-reacted TEMED will focus at the cathode and produce a
more
alkaline pH gradient. Because TEMED is only active as a free
base,
polymerization is inhibited at low pH. BioRad recommends a
final
concentration of 1 to 10 mM TEMED. 22 ?l TEMED/100 ml of gel results
in
1.9 mM TEMED, while 10 ?l TEMED/10 ml of gel results in 8.62 mM TEMED.
Too
much TEMED will increase the tendency of your gel to crack during drying
or
crumble during passive elution due to increased brittleness.)
Initiator concentration
(subscript: These are initiator concentrations that I developed over
the
course of many years of running sequencing resolution RNA gels.
The
decreasing amounts of TEMED result in a consistent polymerization time
and
increased elasticity. The positions of dye markers such as bromphenol
blue
and xylene cyanol remain consistent with published data.)
(subscript: Initiator concentration)(subscript: for degassed gel
solutions
)(subscript: (at Boulder's altitude, degassing is not necessary))
|----------------------------+-------+-----+-----+-----+-----+-----+----->
| | | | | | | | |
| (subscript: % acryl. for | (subs | (su | (su | (su | (su | (su | (su |
| 100 ml gel) | cript | bsc | bsc | bsc | bsc | bsc | bsc |
| | : 4%) | rip | rip | rip | rip | rip | rip |
| | | t: | t: | t: | t: | t: | t: |
| | | 6%) | 8%) | 10% | 12% | 14% | 15% |
| | | | | ) | ) | ) | ) |
| | | | | | | | |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
>-----+-----+------|
| | | |
| (su | (su | (sub |
| bsc | bsc | scri |
| rip | rip | pt: |
| t: | t: | 20%) |
| 16% | 18% | |
| ) | ) | |
| | | |
>-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
| | | | | | | | |
| (subscript: ?l TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| native gel) | cript | bsc | bsc | bsc | bsc | bsc | bsc |
| | : 24 | rip | rip | rip | rip | rip | rip |
| | ) | t: | t: | t: | t: | t: | t: |
| | | 22 | 20) | 18) | 16) | 14) | 13) |
| | | ) | | | | | |
| | | | | | | | |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
>-----+-----+------|
| | | |
| (su | (su | (sub |
| bsc | bsc | scri |
| rip | rip | pt: |
| t: | t: | 8) |
| 12) | 10) | |
| | | |
>-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
| | | | | | | | |
| (subscript: ?l TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| denat. gel) | cript | bsc | bsc | bsc | bsc | bsc | bsc |
| | : 22 | rip | rip | rip | rip | rip | rip |
| | ) | t: | t: | t: | t: | t: | t: |
| | | 20 | 18) | 16) | 14) | 12) | 11) |
| | | ) | | | | | |
| | | | | | | | |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
>-----+-----+------|
| | | |
| (su | (su | (sub |
| bsc | bsc | scri |
| rip | rip | pt: |
| t: | t: | 6) |
| 10) | 8) | |
| | | |
>-----+-----+------|
(subscript: Initiator concentration)(subscript: for non-degassed
gel
solutions)
|----------------------------+-------+-----+-----+-----+-----+-----+----->
| | | | | | | | |
| (subscript: % acryl. for | (subs | (su | (su | (su | (su | (su | (su |
| 100 ml gel) | cript | bsc | bsc | bsc | bsc | bsc | bsc |
| | : 4%) | rip | rip | rip | rip | rip | rip |
| | | t: | t: | t: | t: | t: | t: |
| | | 6%) | 8%) | 10% | 12% | 14% | 15% |
| | | | | ) | ) | ) | ) |
| | | | | | | | |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
>-----+-----+------|
| | | |
| (su | (su | (sub |
| bsc | bsc | scri |
| rip | rip | pt: |
| t: | t: | 20%) |
| 16% | 18% | |
| ) | ) | |
| | | |
>-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
| | | | | | | | |
| (subscript: ?l TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| native gel) | cript | bsc | bsc | bsc | bsc | bsc | bsc |
| | : 26 | rip | rip | rip | rip | rip | rip |
| | ) | t: | t: | t: | t: | t: | t: |
| | | 24 | 22) | 20) | 18) | 16) | 15) |
| | | ) | | | | | |
| | | | | | | | |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
>-----+-----+------|
| | | |
| (su | (su | (sub |
| bsc | bsc | scri |
| rip | rip | pt: |
| t: | t: | 10) |
| 14) | 12) | |
| | | |
>-----+-----+------|
|----------------------------+-------+-----+-----+-----+-----+-----+----->
| | | | | | | | |
| (subscript: ?l TEMED for | (subs | (su | (su | (su | (su | (su | (su |
| denat. gel) | cript | bsc | bsc | bsc | bsc | bsc | bsc |
| | : 24 | rip | rip | rip | rip | rip | rip |
| | ) | t: | t: | t: | t: | t: | t: |
| | | 22 | 20 | 18) | 16) | 14) | 13) |
| | | ) | ) | | | | |
| | | | | | | | |
|----------------------------+-------+-----+-----+-----+-----+-----+----->
>-----+-----+------|
| | | |
| (su | (su | (sub |
| bsc | bsc | scri |
| rip | rip | pt: |
| t: | t: | 8) |
| 12) | 10) | |
| | | |
>-----+-----+------|
(subscript: Initiator concentration)(subscript: for non-degassed
gel
solutions)
|----------------------------+-------+------+------+------+------+------>
| | | | | | | |
| (subscript: % acryl. for | (subs | (sub | (sub | (sub | (sub | (sub |
| 80 ml gel) | cript | scri | scri | scri | scri | scri |
| | : 4%) | pt: | pt: | pt: | pt: | pt: |
| | | 6%) | 8%) | 10%) | 12%) | 14%) |
| | | | | | | |
|----------------------------+-------+------+------+------+------+------>
>------+------+-----+------|
| | | | |
| (sub | (sub | (su | (sub |
| scri | scri | bsc | scri |
| pt: | pt: | rip | pt: |
| 15%) | 16%) | t: | 20%) |
| | | 18% | |
| | | ) | |
| | | | |
>------+------+-----+------|
|----------------------------+-------+------+------+------+------+------>
| | | | | | | |
| (subscript: ?l TEMED for | (subs | (sub | (sub | (sub | (sub | (sub |
| native gel) | cript | scri | scri | scri | scri | scri |
| | : | pt: | pt: | pt: | pt: | pt: |
| | 20.8) | 19.2 | 17.6 | 16) | 14.4 | 12.8 |
| | | ) | ) | | ) | ) |
| | | | | | | |
|----------------------------+-------+------+------+------+------+------>
>------+------+-----+------|
| | | | |
| (sub | (sub | (su | (sub |
| scri | scri | bsc | scri |
| pt: | pt: | rip | pt: |
| 12) | 11.2 | t: | 8) |
| | ) | 9.6 | |
| | | ) | |
| | | | |
>------+------+-----+------|
|----------------------------+-------+------+------+------+------+------>
| | | | | | | |
| (subscript: ?l TEMED for | (subs | (sub | (sub | (sub | (sub | (sub |
| denat. gel) | cript | scri | scri | scri | scri | scri |
| | : | pt: | pt: | pt: | pt: | pt: |
| | 19.2) | 17.6 | 16) | 14.4 | 12.8 | 11.2 |
| | | ) | | ) | ) | ) |
| | | | | | | |
|----------------------------+-------+------+------+------+------+------>
>------+------+-----+------|
| | | | |
| (sub | (sub | (su | (sub |
| scri | scri | bsc | scri |
| pt: | pt: | rip | pt: |
| 10.4 | 9.6) | t: | 6.4) |
| ) | | 8) | |
| | | | |
>------+------+-----+------|
(subscript: Initiator concentration)(subscript: for non-degassed
gel
solutions )
|----------------------------+-----+------+------+------+------+------>
| | | | | | | |
| (subscript: % acryl. for | (su | (sub | (sub | (sub | (sub | (sub |
| 30 ml gel) | bsc | scri | scri | scri | scri | scri |
| | rip | pt: | pt: | pt: | pt: | pt: |
| | t: | 6%) | 8%) | 10%) | 12%) | 14%) |
| | 4%) | | | | | |
| | | | | | | |
|----------------------------+-----+------+------+------+------+------>
>------+------+------+------|
| | | | |
| (sub | (sub | (sub | (sub |
| scri | scri | scri | scri |
| pt: | pt: | pt: | pt: |
| 15%) | 16%) | 18%) | 20%) |
| | | | |
>------+------+------+------|
|----------------------------+-----+------+------+------+------+------>
| | | | | | | |
| (subscript: ?l TEMED for | (su | (sub | (sub | (sub | (sub | (sub |
| native gel) | bsc | scri | scri | scri | scri | scri |
| | rip | pt: | pt: | pt: | pt: | pt: |
| | t: | 7.2 | 6.6) | 6.0) | 5.4) | 4.8) |
| | 7.8 | ) | | | | |
| | ) | | | | | |
| | | | | | | |
|----------------------------+-----+------+------+------+------+------>
>------+------+------+------|
| | | | |
| (sub | (sub | (sub | (sub |
| scri | scri | scri | scri |
| pt: | pt: | pt: | pt: |
| 4.5) | 4.2) | 3.6) | 3.0) |
| | | | |
>------+------+------+------|
|----------------------------+-----+------+------+------+------+------>
| | | | | | | |
| (subscript: ?l TEMED for | (su | (sub | (sub | (sub | (sub | (sub |
| denat. gel) | bsc | scri | scri | scri | scri | scri |
| | rip | pt: | pt: | pt: | pt: | pt: |
| | t: | 6.6) | 6.0) | 5.4) | 4.8) | 4.2) |
| | 7.2 | | | | | |
| | ) | | | | | |
| | | | | | | |
|----------------------------+-----+------+------+------+------+------>
>------+------+------+------|
| | | | |
| (sub | (sub | (sub | (sub |
| scri | scri | scri | scri |
| pt: | pt: | pt: | pt: |
| 3.9) | 3.6) | 3.0) | 2.4) |
| | | | |
>------+------+------+------|
(subscript: Use of more than recommended volume of TEMED will result
in
brittle gels. Note that the volume of TEMED decreases 2 ?l for every
2%
increase in gel concentration in a 100 ml gel stock. )
(subscript: The properties of the gel depend on the concentrations of
APS
and TEMED. Increasing the amount of APS/TEMED leads to an increase
in
chain initiation events and a corresponding decrease in chain
length,
resulting in gel turbidity and decreased gel elasticity. Excess
APS/TEMED
can also oxidize sulfhydryl-containing proteins and result in
altered
buffer pH. Excess TEMED can increase buffer pH, react with proteins
and
alter banding patterns. Excess APS acts as a buffer between pH 8 and 9.
)
(subscript: Reducing the amount of APS/TEMED results in longer
chain
length, lower turbidity and greater elasticity. These are
desirable
properties. Low percentage acrylamide gels are most sensitive to
excess
initiator concentrations, producing soft mushy gels if too much TEMED
is
used. )
(subscript: If concentrations of APS/TEMED are too low, polymerization
will
be too slow and allow air to enter the monomer and result in gels that
are
too porous and mechanically weak. The unpolymerized monomer can react
with
)(subscript: a)(subscript: amino, sulfhydryl and phenolic hydroxyl
groups
of proteins.)
(subscript: Tim Fitzwater)
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