NuPAGE Knowledge Management
By Todd Duell
Voltage (V) = Current (I) x Resistance (R)
Equation 1
Abstract
Watts (W) = Current (I) x Voltage (V)
This knowledge management document has been
prepared for internal training and use. It is the culmination of
information from NOVEX, literature resources, and our
experience qualifying and validating the assay for internal
use. It is not intended to be a comprehensive source of
information for running NuPAGE gel electrophoresis, but
rather a starting point to understand the technical nature of
the assay and how it is used to determine the purity of our
monoclonal antibodies. This document covers basic theory
of electricity, NuPAGE technical information, staining
options, basic monoclonal antibody data analysis,
troubleshooting, recommendations, measurement of
technician performance and qualification, and references.
There is also a reference Excel file, NuPAGE.xls, that should
be used in conjunction with the technician performance and
qualification section.
Electricity
There are two equations that have practical
consequences in electrophoresis:
Equation 2
Resistance is determined by the thickness of the gel
being run and the type of buffer being used. As the run
proceeds, the fast moving, highly conductive Cl- ions in the gel
are gradually replaced by slower moving, less conductive ions
from the running buffer. This causes the resistance of the
system to gradually increase as the run progresses.
The power source used to run the gels will typically have
three settings: constant voltage (V), constant current (I) and
constant watts (W). If constant current is used, both the voltage
and watts will increase as the resistance increases (see figure
1). This could cause a problem with overheating if the run isn’t
monitored or a voltage limit is not set on the power unit.
Unusually high resistance can be caused by frayed
electrode wires, improper electrode connections, or any other
break in the current flow. If this occurs, the voltage will increase
sharply to compensate for the increased resistance. This can
generate enough excess heat to severely damage the gel and
the apparatus. If you are going to run constant current, make
sure you can set the voltage limit on your power supply.
Todd Duell is the Vice President & CIO of Formulations Pro, Inc and has been creating powerful commercial and custom solutions using FileMaker Pro since 1989. He holds an
MBA in Technology Management and has been an Associate member of the FileMaker Solutions Alliance since 1998. Todd may be reached at tduell@formulationspro.com
© 2001 Formulations Pro, Inc. All rights reserved. www.formulationspro.com
If a constant wattage setting is used, the voltage will
increase as current decreases, so the total amount of heat
generated by the system will remain constant (see figure 2).
However, if for some reason the current drops dramatically,
the power supply will try to pump up the voltage to maintain
a constant wattage. If using a high voltage power supply
without the ability to set the voltage limit, this could cause
serious damage to the gel or apparatus.
The recommended method is to use constant voltage
(see figure 3). There are three reasons for this. (1) If
constant voltage is used, both the current and watts will
decrease as resistance increases (see figure 3), thus
improving safety as the run proceeds. (2) The same voltage
setting can be used whether running one or two gels. One
gel provides twice the resistance of two gels. To
compensate, the current for one gel will be half that for two
gels. As a result, the run time will be the same for one or two
gels. When using constant current or watts, the setting must
be adjusted for the number and thickness of the gels being
run. (3) All common electrophoresis power supplies can be set
for voltage, but not all will regulate the current or watts.
Many people would like to run their gels as fast as
possible with the same results. However, the main limiting factor
is heat generation. Excess heat generation can lead to gel
Page 2
distortion and poor band resolution. Figure 4 illustrates what
would happen if we doubled the voltage to halve the run
time. According to Equation 1 (V = I x R), if we double the
voltage, the current must also double. This results in an
approximate fourfold increase in the rate of energy output (5
watts to 21.25 watts). However, since we cut the run time in
half, the total temperature increase is only twofold (10ºC to
22ºC). The effect that this temperature increase will have on
the run depends upon the gel concentration, sample
concentration and solubility, and the apparatus’s ability to
dissipate heat.
As shown, the higher the voltage, the more current,
watts, and consequently heat is generated throughout the run.
For Tris-Glycine gels, the total run time is reduced significantly
from 100 minutes (125V) to 22 minutes (350V) with no visible
effects and only a moderate increase in heat. Obviously the
nature of the sample itself will dictate what temperature it will
tolerate: heat sensitive or native proteins should be run at as
low a voltage as possible to keep heat generation to a minimum.
For running NuPAGE gels with monoclonal antibodies, it
is recommended to use 200 Volts, constant current. The
expected starting current is 110-125mA/gel. The expected
ending current is 70-80mA/gel. With these settings, the run time
will be approximately 35 minutes.
NuPAGE Technical Information
How will excess heat generated by increasing the
voltage affect the quality of the results? Affects may vary, but
in general, properties such as band sharpness, band
curvature, resolution, temperature, and accurate band
migration may suffer. These affects were studied in and
reported by NOVEX in figure 5.
NuPAGE in the presence of a reducing agent (DTT or bmercaptoethanol) is a technique for the separation of
polypeptide subunits according to their molecular weight. The
protocol involves denaturing the protein sample by heating it in
the presence of LDS (lithium dodecyl sulfate) and a reducing
agent. LDS binds to the protein causing it to unfold, whereas the
reducing agent will reduce the intrachain and interchain disulfide
bonds. The LDS will also maintain a constant charge-to-mass
ratio for the protein. This ratio is assumed to be approximately
1.2g LDS/g protein. The binding of LDS by the protein confers a
Page 3
net negative charge and the denatured polypeptide will
migrate through the gel in the presence of an applied electric
field towards the positive electrode or anode (see figure 6).
When running reduced monoclonal antibodies on NuPAGE
under reducing conditions, the IgG molecule will be cleaved
at the interchain disulfide bonds to yield two light chains of
approximately 25Kda and two heavy chains of approximately
52Kda (see figure 7). If samples are not completely reduced,
they may form heterogeneous protein moities that show up
as poorly resolved bands or multiple bands surrounding the
main band.
Sample reducing agents such as DTT or bmercaptoethanol, perform most effectively in a slightly
alkaline pH environment. The NuPAGE Sample buffer has a
pH of 8.45 and allows for the optimal activity of the reducing
agent. The result of effective sample reduction is complete and
efficient cleavage of protein disulfide bonds.
While efficient cleavage of disulfide bonds is desired,
cleavage of peptide bonds should be avoided for the most
accurate results. The NuPAGE sample preparation protocol
minimizes peptide cleavage by emphasizing the importance of
heating the sample for a short period of time (10 minutes) at
relatively low heat (70ºC) and reducing prior to sample loading.
Whereas the traditional Laemmli system of SDS-PAGE is
subject to several problems:
•
The gel is cast at pH 8.7. At this pH, polyacrylamide undergoes gradual hydrolysis, which eventually causes band
distortion and/or loss of resolution. Consequently, the shelf
life of a Tris-Glycine gel is about 1-2 months.
•
The pH of the separating region of the Tris-Glycine gel is
about 9.5 during electrophoresis. At this pH, proteins are
potentially subjected to chemical modifications such as
deamination and alkylation. This may affect subsequent
analyses of proteins following the run.
Page 4
•
The redox state of the gels is not well controlled. This
means that reduced disulfides are more prone to
reoxidation, giving rise to diminished band sharpness and
transfer efficiency, particularly for cysteine containing
proteins.
•
Proteins are subject to cleavage at asp-pro bonds when
heated in the Laemmli sample buffer. Also, reduction of
disulfides is inefficient in the Laemmli sample buffer at pH
8.6. Either condition can lead to artifacts or poor
resolution.
The NuPAGE system also addresses the issues that
impact sample integrity during electrophoresis. The pH
environment is the single most critical parameter in
maintaining sample protein integrity during electrophoresis.
The operating pH of the electrophoresis system (the
cumulative pH of the gel and the running buffers)
immediately regulates the pH environment of the sample
once power is applied to the gel. At a neutral operating pH,
undesirable activity of exposed disulfide groups in reduced
sample proteins is kept to a minimum. As a result, proteins
are kept substantially more intact and unmodified — this
means consistently sharp and unambiguous bands.
However, if the pH is too alkaline or acidic during
electrophoresis, adverse chemical modifications, such as
deamidation, dephosphorylation, alkylation, and oxidation,
may occur to sample proteins. This can negatively impact
the electrophoresis results in several ways. Protein
modification may lead to the weakening of Western blot
signals, decreased yield in protein sequencing, modifying the
mass spectrum of the protein, false and/or multiple bands,
and reduced purity measurements.
The level of protein modification in the NuPAGE system
is minimized because the system pH is neutral. The NuPAGE
Bis-Tris-HCl buffers have a pH of 6.4 and when run with the
MES-SDS buffers, create and effective system pH of 7.
In addition to a neutral pH environment, samples reduced
during sample preparation need to be maintained in their
reduced states. The NuPAGE system prevents reoxidation of
proteins during electrophoresis through the addition of the
Antioxidant to the upper buffer chamber and transfer buffer to
protect other sensitive amino acids such as methionines and
tryptophans. The Antioxidant essentially migrates at the same
speed as the protein to prevent this reoxidation from occurring.
Whereas the LDS will migrate faster than the protein which
allows reoxidation to occur. This prevents split bands and
smearing as the proteins migrate through the gel (see figure 8).
NuPAGE Advantages:
•
Full sample reduction at 70-80ºC for 10 minutes using DTT
prevents denaturation of the protein.
Page 5
•
Using the pH 8.5 sample buffer prevents cleavage of the
asp-pro peptide bonds in the CH2 domain of the heavy
chain. Thus, no “degraded” bands are generated during
sample reduction.
•
Control of the neutral pH running conditions and lower
temperature during the run prevents split bands and
degraded bands. Thus, eliminating confusion and
increasing the purity determination.
•
Since DTT does not co-migrate with the proteins in the
gel, the Antioxidant is used to prevent reoxidation of the
reduced disulfide bonds during electrophoresis. Thus
preventing split bands and smearing as the proteins
migrate down the gel.
•
The Bis-Tris gel with a pH of 6.5 is a more effective buffer
at the neutral pH range. The gel is also more pliable.
Thus, enabling it to withstand higher temperatures,
voltage, and current.
•
Since the Bis-Tris gel is cast at pH 6.4, it will last for up to
one year.
Molecular Weight Estimations
Two factors explain the observation that there are
apparent discrepancies between the size as determined by
gels versus other methods such as sequence analysis and
size exclusion chromatography. First is the amount of LDS
bound to the protein. LDS is employed to disrupt the
secondary structure and give all proteins a constant chargeto-mass ratio. However, as stated by Hjelmeland and
Chrambach, this assumption fails more frequently than is
generally known. The most common deviation from this
assumption is probably a lower than normal amount of LDS.
This is usually caused by under pipetting the Sample buffer due
to the high viscosity of the buffer. A simple solution is to simply
turn up the volume on the pipetman by 50-100%. All else being
equal, mobility would decrease, since the protein would have
less of a negative charge.
The second source of error in molecular weight estimates
is that protein mobility in a gel is more a function of molecular
size (which is a function of both weight and length) than of
molecular mass. It’s generally assumed that LDS proteins all
exist in a random coil form, so the relationship between the
length and mass should be constant. Even assuming constant
charge; if a protein has unreduced disulfide bonds or areas of
incompletely disrupted secondary structure, it cannot unfold to
full length and, it would tend to run faster than expected in a
typical NuPAGE gel.
These deviations from the ideal can combine in every
conceivable way, making it difficult to predict a net effect on
migration rate. Nevertheless, the effects can be large. For
example: BSA will run with an apparent size of 55Kda instead of
67kDa. Furthermore, in smaller proteins a non-ideal region will
have a larger proportional effect than the same region in a large
protein. For example, polypeptides of around 2kDa can give
estimates that are off by a factor of 2 or more from the actual
size.
This is not to say that NuPAGE derived molecular
weights are invalid, just to say that they have limitations. Most
proteins will give estimates within a few percent of their actual
weight by comparing them to appropriate calibration markers.
The closer the marker is to the lane in the gel, the more
accurate the measurement. And possible deviations from “true”
molecular weight do not affect the utility of NuPAGE gels in
Page 6
identification because even unusual proteins, if prepared in
the same way each time should run reproducibly on a given
type of gel.
Finally, care should also be taken in inferring precise
size based on published weights for calibration markers.
Even common proteins may have several slightly different
size estimates reported in the literature, depending upon the
methods of molecular weight measurement.
We use the NOVEX Mark12 Wide-Range Protein
Marker for NuPAGE (see figure 9). The standards used in
this marker behave close to their ideal molecular weight.
Mark12 provides sharp stable bands, which make sample
calibration easy and quick with the help of the densitometer.
The Mark12 contains 12 proteins ranging from 2.5 to 200kDa
for calibration of any gel from 4% to 27% as well as gradient
gels such as the 4-12% Bis-Tris gel used in the NuPAGE
process.
Optimal Sample Loading
A good sample loading strategy is an important part of
optimizing an electrophoresis system to obtain high quality
gel results. This involves determining the correct
concentration of each individual protein, the composition of
the solubilization buffer and the total sample volume per
lane.
The concentration of total sample protein should be
scaled so that each protein of interest is at an optimal load.
This load should be enough to be visualized with the chosen
staining method, but should not exceed the physical capacity
of the gel matrix. This is determined by dimensions such as
thickness, and height and width of the sample well. As a
general rule, the sample should occupy as low a percentage
of the total well volume as possible. Here at BD/PharMingen,
monoclonal antibodies have been optimized to give reproducible
results using densitometry analysis when the protein sample
volume is 10µl and the concentration is between 0.5 to
2.0mg/ml (see figure 10).
If a protein exceeds the capacity that the gel matrix can
accommodate, excessive localized heat is generated as a
heavy overloaded protein band migrates down the gel. In
extreme cases, proteins may migrate between the gel and the
cassette, resulting in a ghost band. Or in less extreme cases,
the band will smear downwards and be poorly defined, giving an
inaccurate
lower
estimation
of
molecular
Page 7
Excessive total sample volume can also cause sample
crossover from lane to lane, causing thin lines or smudges or
duplicate band patterns of the adjacent sample. If a protein is
very dilute, concentration may be necessary. Finally, to obtain
the best results, total sample volume should be kept consistent
from lane to lane.
Staining & Gel Drying
weight and purity. The excessive heat generation may also
distort nearby protein bands in adjacent lanes in a similar
fashion.
Sample buffers have several purposes as previously
stated. The NOVEX reducing agent has sufficient LDS to
denature typical protein concentrations. In order to create an
environment where the role of protein charge is minimized
as much as possible, LDS should be added in excess of the
ration of 1.6g LDS: 1g protein.
Glycerol is present in the NOVEX sample buffer to
increase the density of the sample. This ensures that the
sample will sink to the bottom of the well and facilitates
correct stacking of the proteins.
Once a sample is prepared and diluted properly, it’s
ready to load. The total sample volume in the well will affect
your end result. As a general rule, the well should not be
filled with more than 75% of its total volume. Stacking of
proteins may also be affected if the sample volume is too
high: the larger proteins within the total sample may not
reach the stacking portion of the gel in time to be stacked,
leading to fuzzy and poorly resolved bands.
The staining process used to detect and quantify
antibody purity utilizes G250 Colloidal Coomassie staining
chemistry. This process provides nanogram-level detection of
proteins and crystal clear backgrounds without a harsh chemical
de-staining process. The method is based on the colloidal
properties of Coomassie Blue dye created in aqueous or
methanol solutions containing inorganic acids and high salt
concentrations. The free dye in solution is greatly reduced due
to the hydrophobic effect, resulting in low background staining
and high affinity binding of the dye to the proteins fixed in the
gel.
This method of staining is five times more sensitive than
the traditional R250 Coomassie technique. With G250, 10ng of
protein can be detected on standard 4-12%, 1.0mm thick TrisGlycine gel. The key to the process however lies in the
downstream capabilities afforded by this method. The 10-minute
ethanol and acetic acid rinse removes the SDS and opens up
the gel pores to enhance the staining capabilities of the dye.
The DI Water destain process does not remove the dye from the
protein. As a result, you are left with a crystal clear background
that enables densitometry quantitation of purity to be far more
accurate and consistent (see figure 11).
Page 8
There are trade-off’s in the process between
traditional R250 and Colloidal G250 as shown in figure 12. If
you don’t need to perform densitometry analysis, the
recommendation is to use traditional Coomassie R250
techniques. However, if densitometry analysis is required,
there is no better choice than Colloidal G250.
Once you’ve stained your gels, it is imperative to dry
them appropriately for long term storage and use. If the gels
are not treated properly, they will eventually crack and
shrivel up — rendering them useless. The drying of gels
between two sheets of transparent cellophane after
electrophoresis and staining is a convenient way to preserve
and permanently store the gels. Gel drying can become a
frustrating experience if your gels crack during the process,
since a single gel can represent hours of time invested into
the sample preparation, running, and staining of the gel.
Gels should be handled with care when removed from
the cassette to ensure that all the edges are as straight as
possible. Even slight nicks and tears can act as starting
points for large cracks to develop. One recommendation to
limit this from happening is to remove the loading wells and
the foot of the gel with the use of a gel knife. If physically
handling the gel, the bottom of the gel with a higher
concentration of material is stronger than the stacking portion
(top) of the gel with a low percentage of material.
Immediately after removing the wells and the foot of the
gel, the gel should be equilibrated in a drying solution. The
drying solution contains alcohol to prevent excessive swelling of
the gel as well as glycerol, which helps to regulate the rate of
drying. Therefore, preventing cracking. Since the alcohol can
remove stain from the proteins in the gel, it should not be
soaked in this solution for more than 10 minutes.
The gel is then placed between two pieces of cellophane
for support to help prevent the gel from shriveling up. To prevent
cracking, it is critical not to allow trapped air between the
cellophane sheets and the gel. There are several methods to
remove the trapped air from under the cellophane sheets. First,
Page 9
don’t be afraid to use too much DI Water when preparing the
cellophane sheets and the gel on the platform — more is
better. Second, if air bubbles are still trapped between the
cellophane sheet and the gel, gently press them out to the
edge of the gel with your finger. Third, use a transfer pipette
to suck the bubble away from the gel.
Once you have the gel prepared for drying, it will take
approximately 24 hours for a passive drying system. There
are several types of faster drying systems available such as
vacuum and convection ovens, but one should be aware of
the pitfalls involved in using these devices. While drying the
drying period is shortened, the rapid rate at which water is
removed from the gel increases the chances of cracking.
Another concern exists when using a heated gel dryer. The
rate of the heat flowing across the gel can be inconsistent
from one drying session to the next. Thus making it difficult
to determine the exact length of time for drying. Excessive
heat can also cause the cellophane to shrivel up.
interchain disulfide bonds, leaving a set of 52kDa (heavy chain)
and 25kDa (light chain) components (see figure 13). The IgM
antibody is made up of a pentamer (5) of Ig molecules held
together by a J chain (see figure 14). The J chain is not cleaved
during reduction. Therefore, the reduced heavy chain for an IgM
will actually have two heavy chain subunits with a combined
size of approximately 100kDa (see figures 13 and 14). IgA can
come in the form of a dimer or a monomer. In the event that the
molecule is a dimer, the reduced results may be similar to an
IgM molecule. Otherwise the reduced results will be similar to
an IgG molecule.
Data Interpretation
The data interpretation that is provided covers the
majority of the typical scenarios for monoclonal antibodies
purified by affinity chromatography: normal IgG and IgM/IgA,
bovine Ig, mycoplasma, split or double bands, 30kDa bands,
degraded antibody, HPCD issues, and gel “smiling”. The
reasons for all variations are as diverse as the problems that
may cause them. I would hesitate to provide an exact cause
in some cases without specific investigation. Therefore, only
a list of possible causes will be given for each of the cases
shown below.
In some cases the IgM’s J chain will be broken during
reduction and/or electrophoresis. If this happens, there will be a
mixture of 100kDa, 52kDa and 25kDa bands on the gel. All
three should be included in the purity of the product unless you
are aware that the 52kDa band belongs to an IgG molecule (see
figure 15).
IgG, IgM, and IgA Identification: As previously mentioned,
during reduction, the IgG antibody will be cleaved at the
Page 10
weight bands have a strong correlation with this bovine Ig
complex. The bovine Ig bands are not included in the purity of
the product.
Bovine Ig (gamma globulin) Identification: In most cases, 1%
to 10% fetal bovine serum (FBS) is fed to the tissue culture
to aid in stimulating the cells to grow and secrete antibody.
Some of the gamma globulin complex will bind to the affinity
resin during the purification process and elute with the
product. In the cases where there is a relatively high ratio of
bovine Ig to antibody, there will be three distinct higher
molecular weight bands that appear on the gel (see figure
16). From previous IEF studies with purified FBS, we know
that the pI is 4.8 to 5.2 and that the three high molecular
Mycoplasma Identification: We are not actually identifying
mycoplasma with NuPAGE because mycoplasma does not bind
to affinity chromatography resin. What we are identifying is the
protein that is left behind from the presence of mycoplasma in
the tissue culture. Mycoplasma secretes a 95 to 105kDa dimer
protein that binds to the Fab region of the light chain
nonimmunologically (see figure 17). Under reducing conditions
the mycoplasma protein is broken down into two components —
two 43kDa subunits and two 10kDa subunits (see figure 18). If
these appear on the gel it is usually a clear indication of
mycoplasma contamination. There is also some speculation that
when the PCR-based mycoplasma and Western blot assays are
negative that this problem is caused by protease activity. Either
way, purification protocols are unable to solve the contamination
or degradation problems. Fixing the tissue culture is the only
course of action that can solve the problem. The 43kDa and
10kDa bands are not included in the purity of the product.
Page 11
electrophoresis, different molecular weights for each heavy or
light chain component, or even the molecule being a different
size that the Bovine Ig heavy or light chain. Which will then
show both sets of bands. In any case, split bands do not mean
that the antibody is of poor quality. In most cases both bands
will be included in the purity of the sample.
Split Heavy Chain and Light Chain Identification: This is
identified by two or more heavy or light chain bands that are
close together (see figure 19). They are not to be confused
with a 30kDa band (see below for more details). It could
arise from a variety of sources such as reoxidation during
30kDa Band Identification: The 30kDa band is slightly higher
than the light chain band (see figure 20). This is not to be
confused with a split light chain where the bands are very close
together. The 30kDa band can come from a variety of sources
such as reoxidation during electrophoresis, degradation, and
protease activity. However, it is usually associated with a
separate protein secreted from the hybridoma’s fusion partner.
This protein may need to be present for the activity of the
protein. However, unless its origin is known (i.e. the fusion
partner is a know secretor), it will not be included in the purity of
the sample.
Page 12
of the gel. As a result, it sucks in the adjacent lanes (see figure
22). This problem is more pronounced with higher
concentrations of HPCD. If you need to run a sample with
HPCD, it is highly recommended to leave the adjacent lanes
open.
Degraded Antibody Identification: Any bands that are below
the light chain are considered degraded antibody. Whether it
is from protease activity, mycoplasma, degradation during
reduction and electrophoresis, these bands are not included
in the purity of the sample (see Figure 21).
Gel “Smile” Identification: As mentioned above, the proteins will
migrate at different rates as you move away from the center of
the gel. This phenomenon is called gel “smiling” (see figure 23).
It does not affect the purity calculations, but it will affect
molecular weight estimations the further away the sample is
from the standard.
HPCD Identification: HPCD is essentially a big sugar
molecule. During electrophoresis, HPCD pulls the water out
Page 13
called two-population, dependent Hypothesis testing can be
used to measure their capability to provide accurate data.
Step 1: State the question and Null Hypothesis.
Are the technician’s values the same the approved
values?
Ho = the approved values. H1 = the technician’s
values.
Technician Qualification and Performance
Measurement
How do you measure the qualification and
performance of your technicians and trainers to ensure
quality and consistency not only for an individual technician
but also within and between groups? What qualification and
performance measures should be used? These
measurements can be performed with a variety of simple
statistical techniques explained below.
Technician Densitometer Qualification
Technicians can be qualified to use the densitometer
for providing purity quantitation by measuring their test
results against an “approved” set of qualified results or
“standards”. These standards should contain a normally
distributed variety of extreme results such as high
background and extra and/or contaminant bands. The more
results the technician scans, the more accurate the results.
There should be at least 30 results to be statistically
significant and not have to adjust for additional degrees of
freedom. With the technician’s results, a statistical measure
Ho=H1
Ho≠H1
Step 2: Determine the Confidence Interval (a) and select the
Test Statistic (s or t).
a = 0.05 (95% Confidence Interval)
z = 1.96 (2-tailed test. The z statistic is used because
the standard deviation of the approved values
is known and 30 values will be measured.)
Step 3: State the Rejection Hypothesis for a 2-tailed test.
Reject Ho if z >1.96
Reject Ho if z >-1.96
Step 4: Calculate the z value.
z=
d
Sd ÷ n
Equation 3
Page 14
Where:
S
d
=
2
(Â d) ÷ n˘˙˚
 d 2 - ÈÍ
Î
n -1
d = Approved Values — Technician’s Values
Step 5: State the conclusion.
Do not Reject Ho if z < 1.96 or z >-1.96.
Reject Ho if z >1.96 or z <-1.96.
If the Null Hypothesis (Ho) is rejected, the technician’s
values are not the same as the approved or standard values.
The trainer will then need to investigate the reason why they
are not the same. If the Null Hypothesis is not rejected, the
technician’s results are the same as the approved or
standard values and the technician is approved to perform
the analytical testing. The technician should not be allowed
to perform the analytical testing until they meet the
qualification standards. For further information, see the
Densitometer Analysis Qualification worksheet in the
NuPAGE.xls file.
Technician Long Term Performance Monitoring
Once the technician has been qualified to perform the
analytical quantitation, a long-term strategy has to be in
place to ensure that consistent results are reported. Since
one of the biggest factors in providing consistent and
accurate data is the background staining of the gel, this will
be the easiest value to monitor.
The method to monitor the long-term performance is call
Statistical Process Control (SPC). A process is considered
capable if it has a process distribution whose extreme values fall
within the upper (UCL) and lower control limits (LCL). The LCL
value would be the background associated with a gel that has
been run through the electrophoresis process without samples
or the staining process (approximately 0.048 OD). The UCL
value would be the average of several gels that have been run
with samples and stained (approximately 0.052 OD). As a
general rule, most values of a process should fall within ±3s of
the mean. Hence, if a process is considered capable, the
difference between the upper and lower specifications must be
greater than 6s. This is called the Process Capability Ratio
(Cp).
Cp = (UCL — LCL) ÷ 6s
Equation 4
Critical Value:
1. If Cp >1.0, the tolerance range is greater than the range
of actual process outputs and the process will produce
products within their allowable tolerances.
2. If Cp <1.0, the tolerance range is less than the actual
process outputs and the process will produce products
outside their allowable tolerances.
The process is considered capable only when Cp is greater
than the Critical Value (i.e. 1.0) and the process distribution is
centered on the mean value of the tolerances. For example, if
the process distribution is closer to the UCL, the likelihood that
defects will occur is higher. Thus, we need to compute the
Process Capability Index (Cpk) that measures the potential for
the process to generate outputs relative to either the UCL or
LCL.
Page 15
Equation 5
Cpk = Minimum of [(Mean — LCL) ÷ 3s, (UCL —
Mean) ÷ 3s]
In this equation, we take the minimum of the two ratios
because it represents the worst case situation:
•
•
If Cpk > 1.0 and Cp > 1.0, the process is considered
capable.
If Cpk < 1.0, the process average is close to one of the
tolerance limits and is generating defective output.
Cpk will always be less than or equal to Cp. When Cpk
equals Cp, the process is centered between the UCL and
LCL and hence the mean of the process distribution is
centered on the mean of the specifications.
If you graph these results using Excel, what you will
see is a graph similar to figure24. It’s always good to graph
the results so you can visually monitor and stop the process
if there are any emerging trends that could result in
problems. For further information, see the SPC
worksheet in the NuPAGE.xls file.
Measuring Group Consistency
The way to measure group performance (for background
OD consistency) of two or more technicians is through ANOVA
(analysis of variance) analysis. In performing ANOVA, you
determine what part of the variance you should attribute to
randomness and what part you can attribute to other factors.
ANOVA does this by splitting the total sum of squares into two
parts; a part attributed to differences between groups and a part
due to random error or random chance. For ANOVA to work,
there are three assumptions that must be true:
•
•
•
The data must be Interval (zero has no meaning) or Ratio
(zero has meaning)
The data must be normally distributed.
All the variances (between technicians) are equal.
Variance Testing
Before spending the time to perform all the ANOVA
calculations, every possible combination of variances should be
tested. You will need to know the mean, standard deviation and
sample size for each technician. The sample sizes don’t have to
be the same. You will also need access to an F distribution
statistical table to perform this function.
Step 1: State the question and Null Hypothesis.
Is the variance for technician 1 different than technician
2?
Ho: s21 = s22
H1: s21 ≠ s22
Page 16
Step 2: Determine the Confidence Interval (a).
Ho is not rejected, you can proceed with ANOVA analysis
because the variances are the same. Remember that all
possible combinations of technician results must be
tested before including them in ANOVA analysis.
a = 0.10
Step 3: Select the Test Statistic (F).
2
1
2
2
F = S ÷ S
Equation 6
S21 is always the larger standard deviation value.
If you don’t have access to an F table, I would
recommend using the Excel F-Test Two-Sample for Variance
built in function to perform you calculations. For further
information, see the Background OD ANOVA worksheet in the
NuPAGE.xls file.
S22 is always the smaller standard deviation value.
Step 4: State the decision rule for a 2-tailed test (positive
values only).
Reject Ho if F > (df1 ÷ df2) = (n1 –1) ÷ (n2 –1)
n1 = the sample size numerator value for the S21
standard deviation
n2 = the sample size numerator value for the S22
standard deviation
Use the 0.05 F table for a = 0.10 to find the F value.
Step 5: State the conclusion.
Reject H0 if: S21÷ S22 > (n1 –1) ÷ (n2 –1)
Do not Reject H0 if: S21÷ S22 < (n1 –1) ÷ (n2 –1)
If Ho is rejected, you cannot perform the ANOVA
analysis because the variances are not the same. If
Page 17
ANOVA Testing
The easiest way to perform this calculation is to use
Excel’s Data Analysis function. To understand the meaning
of the F calculation that Excel will give, ANOVA is calculated
as follows:
Step 5: Calculate F and make the decision.
Step 1: State the question and the Null Hypothesis
Ho: µ1= µ2= µ3…
H1: Not all µ’s are equal
Step 2: Select the level of significance.
a = 0.05
SSTR = Â nj
j=1
Step 3: Select the test statistic.
F = Between Sample Variances ÷ Within Sample
Variances
K
n
SSE = Â Â
j =1 i =1
(x -x )
(x
j
2
)
ij - x j
Where:
Step 4: State the decision rule.
F > (K - 1) ÷ (N – K)
2
K
Equation 7
K = the number of technician’s being compared
N = the total number of data points being
compared
Results are taken from an F statistic table where (K-1)
is the numerator value and (N-K) is the denominator
value.
j = column number
i = row number
nj = sample size of the column
x
j
= mean of the column
x = mean of the means
Reject Ho if (K-1)÷(N-K) > MSTR÷MSE
Page 18
If you Reject Ho then your group is not providing
consistent data. If you do not Reject Ho then your
group is providing consistent data.
These calculations can get pretty nasty as you
compare more and more data. In this case, I strongly
recommend that you use the built-in ANOVA-Single Factor
calculations provided by Excel. For further information, see
the Background OD ANOVA worksheet in the NuPAGE.xls
file.
•
Has previously met all the statistical qualification for
accuracy and consistency.
•
Able to explain the technical details of the analytical process
for running gels as well as evaluating and monitoring the
qualification and performance of the technicians.
•
Able to correctly answer questions about the theory, routine
technical and troubleshooting issues.
•
Is able to troubleshoot raw materials and equipment
problems.
•
Has demonstrated the ability to correctly interpret the
analytical results.
•
Is capable of communicating all of the training information
with the technicians during the training process.
•
A high percentage of technicians are able to pass the
rigorous qualification process after receiving training.
•
Has demonstrated superior communication abilities.
Troubleshooting & Recommendations
Trainer Certification
There is frequently a question about the difference
between who is qualified to run NuPAGE and who is
qualified as a NuPAGE trainer. Some of the differences that
separate a trainer from a technician include:
•
Why is Bis-Tris used as the gel buffer as opposed to
Tris, which is used in the Tris-Glycine system?
Bis-Tris has a pKa of 6.5, so it is a useful buffer in the
neutral pH range. Tris, on the other hand has a pKa of 8.1
and is not an effective buffer below 7.4.
Page 19
•
Is there SDS in the NuPAGE Bis-Tris gels?
•
No. Sufficient denaturing conditions are created by the
LDS in the sample buffer.
•
•
The NuPAGE Bis-Tris gels are composed of a different
matrix, and as a result are slightly more pliable. They are
also more resistant to heat during the electrophoresis
process.
Why can’t I run NuPAGE under native conditions?
The high ionic strength of the NuPAGE system provides
good stacking of LDS denatured proteins but creates too
much heat for many native proteins. Also, at neutral pH,
many native proteins do not have enough of a charge on
them to migrate.
•
Is the stacking gel shorter on the NuPAGE gels in
comparison to other gels?
Yes, by about 20%. This still allows for complete stacking
of proteins while increasing the length of the separating
gel.
•
What are the storage conditions for the NuPAGE
gels?
The life expectancy of the gels is 1 year if stored in the
cold room (2-8ºC). However, the performance of the gels
will not suffer if they are left out at room temperature
temporarily or even over the weekend.
Why is the NuPAGE Sample Buffer pH 8.45 if the claim is
that a neutral pH environment is better for
electrophoresis?
For optimal sample preparation in all SDS-PAGE protocols it
is best to denature and reduce the protein disulfide bonds
under slightly alkaline pH conditions. Sample reduction at
the pH of the NuPAGE Sample Buffer (pH 8.45) allows for
maximum activity of the reducing agent. Also, heating should
be controlled to mild conditions (70ºC) for a short period of
time (10 minutes). After the controlled sample preparation
step, the neutral pH in the electrophoresis environment
keeps the reactivity of the disulfide groups on reduced
sample proteins to a minimum.
Is there a stacking gel on the NuPAGE gels?
Yes, it is a 4% stack.
•
Why are the NuPAGE gels more pliable that other types
of gels?
•
Why is the NuPAGE Sample Buffer so difficult to
pipette?
The NuPAGE Sample Buffer is a 4X concentrated solution.
The presence of sucrose also increases the viscosity of the
NuPAGE Sample Buffer. There are two recommendations to
deal with this issue. First, by brining the Sample Buffer to
room temperature (25ºC) either by storing it at room
temperature, placing it in a water bath, or an incubator, will
make the Sample Buffer easier to manage. Second, when
Page 20
pipetting the solution, increase the micrometer setting by
50 to 100% to ensure that the correct quantity of Sample
Buffer is used.
•
Can other reducing agent be used other that DTT —
such as b-mercaptoethanol?
The NOVEX Reducing Agent is 500mM Dithiothreitol 10X
concentrate in a stabilized, liquid form. The advantage to
using DTT from NOVEX is that it is ready to use. Thus,
there is no need for preparation from powder. DTT is also
far safer than b-mercaptoethanol from a hazardous
material stand point.
b-mercaptoethanol is compatible with the NuPAGE
system. The choice of reducing agent is usually a matter
of preference or habit. In any case, the sample
preparation protocol should still be followed whether
using DTT or BME. However, it’s always a good idea to
leave the reducing agent out of the sample until just
before the run. To prevent exposure to a hazardous
chemical BME should be prepared and used under a
fume hood.
•
•
The antioxidant is intended to keep sample proteins which
have already been reduce in the sample preparation step
from reoxidizing during electrophoresis. The antioxidant itself
is not efficient to reduce disulfide bonds.
If antioxidant is used when no previous sample reduction is
performed, the end result will be partially reduced bands with
susbstancial background smearing traveling down the lane.
•
If DTT is added to the sample during sample
preparation, shouldn’t that be sufficient to keep the
samples reduced during electrophoresis? What is the
exact purpose of the Antioxidant?
In the neutral pH environment, DTT (and BME) do not comigrate through the gel with the sample. While the disulfide
bonds are less reactive at neutral pH and therefore less
likely to reoxidize than in a higher pH system, some
reoxidation may occur during the run, resulting in slightly
diffuse bands. By adding the Antioxidant, which does comigrate through the neutral pH during the gel, proteins are
kept from reoxidizing. Additionally, the Antioxidant protects
sensitive amino acids in addition to cysteine groups such as
methionines and tryptophans from oxidizing. Thus, band
sharpness will be enhanced.
How fresh does the Reducing Agent have to be in
order to obtain complete reduction?
The Reducing Agent should be added to the sample
within one hour of loading onto the gel. As long as the
Reducing Agent (DTT) is used within the expiration date,
the results are reproducible. Since the Reducing Agent is
purchased as part of a kit for 10 gels, when the last gel is
used please throw away all the components of the kit to
ensure that expired materials are not being used.
What happens if I forgot to reduce my sample? Will the
antioxidant create reducing conditions?
•
Can I run reduced samples with non-reduced samples
on the same gel? If so, do I still add the Antioxidant to
the Running Buffer?
Page 21
For optimal results, it is not recommended to run reduced
and non-reduced samples on the same gel. However, if
you choose to mix reduced and non-reduced samples on
the same gel, they should not be placed in adjacent
lanes. The reducing agent may have a carry-over affect
on the non-reduced sample if they are too close in
proximity. Also, if you are running non-reduced samples,
you should not add the Antioxidant to the upper buffer
chamber. The Antioxidant may slightly reduce native
proteins.
•
How fresh should the Antioxidant be when it is added
to the upper buffer chamber?
It should be used within 30 minutes of preparation.
Substantial loss of activity has been observed if it is used
two hours after preparation. The proteins may exhibit
signs of reoxidation.
•
Can the NuPAGE Antioxidant be used in other gel
systems?
No. It’s not efficient at the higher pH’s of other systems.
Why can’t I filter the G250 Colloidal Coomassie dye
solution before I use it?
The colloidal dye particles that are formed in solution will be
retained on the filter.
•
Why does the G250 stain need to be prepared fresh
before I use it — can’t I just mix all the stain
components together to make a final stock solution?
The G250 stock staining solution is stable for prolonged
periods of time, but should be agitated thoroughly before it is
used. The methanol, ammonium sulfate, and phosphoric
acid solution should not be prepared in advance as a result
of evaporation of the methanol. The reduction in methanol
due to evaporation will reduce the rate of staining in the
protein.
How poor will the results be if I forgot to add the
Antioxidant to the upper buffer chamber?
Some bands may be slightly fuzzier, diffuse, and/or split
than if the Antioxidant is used due to some reoxidation
during electrophoresis. There will be differences from
protein to protein.
•
•
•
If methanol can destain the gel, why is it used in the
staining process?
The inclusion of 20% methanol into the G250 staining
solution shifts the equilibrium form the colloidal form to the
molecularly dispersed dye form, facilitating diffusion into the
gel matrix with resultant faster and more complete protein
staining throughout the entire cross section of the gel. Higher
concentrations of methanol, as in conventional R250 staining
protocols result in heavy background staining, and in
addition, strongly interfere with the formation of protein-dye
complexes. The formation of stable protein-dye complexes is
crucial for high staining sensitivity whereas the formation of
colloidal dye particles is crucial for avoiding background
staining. Having a clear background and thoroughly stained
Page 22
proteins is critically important for accurate and
reproducible densitometry analysis.
•
What is the purpose of the ammonium sulfate in the
staining solution?
The formation of colloidal dye particles depends on the
concentration of ammonium sulfate in the staining
solution and determines the background staining. The
optimal concentration is 8% ammonium sulfate to keep
the background of the gel clear.
•
•
I need to analyze a large molecular weight protein —
what is the largest protein that can be separated by
the NuPAGE Bis-Tris gel?
What is the purpose of the phenol red in the Sample
Buffer?
Phenol red is used as a true ion-front tracking dye for the
very small pore-sized gels. Phenol red is smaller than
Coomassie G250. In high percentage gels, molecules of this
size are resolved on the basis of size, such that phenol red
runs with the ion front while G250 can run as much as 2cm
behind the ion front. It is generally a more useful tracking
dye than G250.
What is the purpose of the initial destain step using
acetic acid and methanol?
The methanol opens up the gel matrix pores, which
allows the acetic acid to facilitate the removal of LDS
from the protein and gel. This step will enhance the
staining of the Coomassie G250 stain.
•
process, we have qualified a10µl sample at 0.5 to 2.0mg/ml
as the optimal loading volume and concentration. If you load
too much volume or too high a concentration, it will overload
the gel. As a result, the entire lane may be smeared and/or
overlap into the adjacent lanes.
•
Why is LDS (lithium dodecyl sulfate) used in the Sample
Buffer rather than SDS (sodium dodecyl sulfate).
SDS in the 4X Sample Buffer tends to precipitate out of
solution and to make the buffer more viscous and difficult to
pipette. LDS is much more soluble even at cold room
temperatures (2-8ºC).
The largest size the Bis-Tris gel can resolve is 400kDa
(reduced Laminin). Proteins larger than this will not be
able to exit the stacking portion of the gel.
•
How much sample can I load into the well?
No more than 75% of the total well volume should be
loaded. Preferably, the smallest possible volume should
be used to achieve the sharpest bands. For the NuPAGE
Page 23
References
1. www.invitrogen.com. 3/23/2001.
2. Hjelmeland, L.M. and Chramback, A., Electrophoresis
1981, 2, 1-11.
3. Sallantin, M., Huet, J., Demarteau, and Pernollet, J.,
Electrophoresis 1990, 11, 34-36.
4. Moos, M., Journal of Biological Chemistry, Vol. 263, pg.
6005-6008.
5. NuPAGE Electrophoresis System Technical Guide.
Version EP049, 1996.
6. Davagnino, Juan, et al, Acid Hydrolysis of Monoclonal
Antibodies. Journal of Immunological Methods, 1995,
185, 177-180.
7. Kubo, Kanenobu, Effect of Incubation of Solutions of
Proteins Containing Dodecyl Sulfate on the Cleavage of
Peptide Bonds by Boiling, Analytical Biochemistry, 1995,
225, 351-353.
8. Abstract from the Ninth Symposium of the Protein
Society, July 1995. Protein Science 4 (Suppl.2):161.
9. Novex News, The Power of Electrophoresis, Volume 2,
Issue 4, 1993.
11. Novex News, Sensitive, Easy, Colloidal Coomassie Staining
Kit, Volume 4, Issue 2, 1994.
12. Novex News, NuPAGE: Sample Stability ~ A Crucial
Element in Superior Electrophoresis Results, Volume 6,
Issue 1, 1996.
13. SOP: MF/5162, Performing NuPAGE Gel Electrophoresis
Using a Precast SDS-PAGE Gel and Electrophoresis Cell
From Novex, Revision 3, 10/16/2000.
14. Chait, B.T. and S.B.H. Kent, Science, 257: 1885-1894 1992.
15. Woods, E.F., Himmelfarb, S. & Harrington, W.F., J. Biol.
Chem., 238, 2374 1963.
16. Brown, J.R., Fed Proc., 34 591 1975.
17. Jolles, P., Agnew, Chem. Intl. Edit., 8, 227 1969.
18. Neuhoff, et al; Improved staining of proteins in
polyacrylamide gels including IEF gels with clear background
at nanogram sensitivity using Coomassie Brilliant Blue G250 and R-250. Electrophoresis 1988, 9, 255-262.
19. NOVEX, Colloidal Coomassie Stain Instructions.
20. NOVEX, NuPAGE Electrophoresis System Instructions.
21. NOVEX, Precast Gel Instructions.
10. Novex News, Molecular Weight Estimation and SDSPAGE, Volume 2, Issue 1, 1992.
Page 24
22. Berk, Kenneth and Carey, Patrick. Data Analysis with
Microsoft Excel. Duxbery Press 1998.
23. Alexander, Angelia, et al. Identification of a Mycoplasma
Protein
Which
binds
Immunoglobulins
Nonimmunologically. Infection and Immunity, June 1991,
pg. 2147-2151.
24. Maddsen, Randall, et al. Species-Specific Monoclonal
Antibody to a 43,000 Molecular Weight Membrane
Protein of Mycoplasma pneumoniae. Journal of Clinical
Microbiology, October 1986, pg. 680-683.
25. Pawar, Anil. Applied Managerial Statistics — MGT/572.
University of Phoenix. Masters in Business
Administration, Technology Management, 2000.
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