Methods and reagents: Eliminating banding artifacts from SDS-PAGE

Methods and reagents is a unique monthly column that highlights current
discussions in the newsgroup bionet.molbio.methds-reagnts, available on the
Internet.  This month's column discusses how to avoid some artifacts when
running SDS-polyacrylamide gels, and some other tips for reducing the cost
of lab supplies are given as well.  For details on how to partake in the
newsgroup, see the accompanying box.

Frank Taddeo (taddeof@jeflin.tju.edu) was having problems with extra bands
appearing on his gels when performing sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). Several dark bands were seen migrating at
approximately the same position as a 58-68 kDa protein throughout all the lanes
of the gel after it had been silver stained.  Although the bands were not
interfering with the results of his particular experiments, they continued to
be an obvious, but bothersome contaminant, even when high-end reagents such as
ultra-pure acrylamide, milli-Q water and analytical-grade glycine were used.
Other netters have also complained that they had a similar problem, and having
been unable to eliminate the extra bands, they have simply put up with them.

The usual suspects
******************
Proteolysis or BSA?
In earlier postings about the same problem, someone suggested that unexpected
bands migrating at this molecular weight could be caused by proteolysis of the
test samples, and that perhaps only fragments of the expected protein are then
seen.  However, this could not explain why the bands were found in all lanes
including those containing control samples without protein added.

Others thought that the bands were owing to some other protein, perhaps bovine
serum albumin (BSA), which is known to be 68 kDa and had been added to the
sample.  Alternatively, one of the reagents used for preparing the gel could
have been contaminated.  A low grade glycine stock could be contaminated with
protein from which it was derived or with something else during the
manufacturing or handling processes.

Mercaptoethanol.
One netter said that bands of this size are known to be caused by contaminants
found in beta-mercaptoethanol (2-ME) and suggested that the solution be
filtered through a 0.2 um pore size filter before use, or stored at -20 degrees
C then centrifuged to remove any precipitates before use. [1] Others thought
that the SDS, which is added to the gel, is what is causing the bands, but one
netter claimed that an artifact band from SDS is known to occur at a lower
position on the gel corresponding to about 10 kDa.  The use of 2-ME in loading
buffer and the inclusion of SDS within the gel are meant to keep disulfide
bonds in the reduced state and thus they prevent the re-naturation of proteins
during the gel run. They are therefore necessary components of the procedure.

However, one trick suggested by netters to eliminate any unwanted artifacts
caused by 2-ME itself is to remove it after reducing the proteins.  This is
done by treating the samples with iodoacetamide to cap the free thiol groups by
alkylation after the denaturation step. Essentially, this prevents the
renaturation of the proteins and the 2-ME can be washed away or diluted out
before the samples are run on the gel. [2] The drawback of this, however, is
that the sample requires much more handling and there is the risk of losing
much or all of the precious sample.

Alternatively, the 2-ME can be replaced with dithiothreitol (DTT),
tris-(2-carboxyethyl)-phosphine (TCEP) or sodium 2-mercaptoethanesulfonate
(MESNA).  TCEP and MESNA have the added advantage that they are water-soluble
and odorless.  [3,4]

It has been known for some time that artifacts can be created by use of 2-ME,
but that 2-ME is probably not what causes the bands. It has been observed that
a 68 and 54 kDa size bands occur when 2-ME is run on a gel by itself, and that
occasionally a doublet at 68 kDa and two other bands of an apparent molecular
weight lower than 54 kDa are seen.  Reduction of contaminating proteins or
other components within the gel itself was likely to blame. [5]

Keratin.
The troublesome bands might be derived from human keratin, which could come
from sloughed human squamous epithelial cells, bits of skin, hair or finger
nails, or dandruff, which can contaminate the entire lab in the form of
microscopic dust particles, eventually making their way into various SDS-PAGE
reagents, including the 2-ME. However, the dust could also be sheep keratin
from wool fibers of clothing.  Keratin has also been found to be a common
contaminant on western blots when samples have been prepared under reducing
conditions. In one case, exposure of the upper buffer to the experimenter's
right hand showed artifactual bands within the gel, while an unexposed one did
not.  However, the results of that experiment might be suspect as no left-hand
control was performed. [6]

Although probably present in all polyacrylamide gels, the artifactual bands are
usually seen when the gel is silver stained, and in general not noticed when
the gels are stained with Coomassie Brilliant Blue (CBB). This is because
silver staining is a much more sensitive method.

Mean, clean, protein machine
****************************
Those who have cured themselves of the problem completely, eliminated the extra
bands with meticulous handling technique, which includes wearing latex gloves
whenever touching any substance or piece of apparatus involved in the
electrophoresis setup, and the scrupulous cleaning of gel boxes, glass plates,
combs, etc., and sonicating the combs.  One consideration is the type of
detergent used to clean the glass plates before pouring the gel. For example,
Contrad [R] NF available from Curtin Matheson Scientific, which contains sodium
dodecylbenzenesulfonic acid and potassium hydroxide, and Micro Detergent from
International Products, Burlington, NJ, USA have been found to eliminate some
of the background problems associated with silver staining of polyacrylamide
sequencing gels.  Any amount of SDS or other detergent left on the plates could
cause background or other problems and the glass plates should be rinsed
thoroughly every time they are cleaned. [7]

Unfortunately for Mr Taddeo, after deciding that the bands were most probably
caused by an older bottle of 2-ME used for the sample buffer, fresh bottles of
different grades of 2-ME from several companies were tried with the same
result, indicating that the proteins or whatever caused the bands were not
present in the older reagent. Regardless of the source, caution should be
observed and proper controls should be done when characterizing proteins by
SDS-PAGE and western blotting as false bands and cross-reactivity of antibodies
to them can be misleading. [8,9]

Spin Doctor
***********
Now that we are fully into the digital age and vinyl records are a thing of the
past, what should you do with that old LP record player that has been gathering
dust in the closet? How about moving it into the lab and converting it into a
shaking incubator? `Huh???', you say...but this is precisely what Bob Horton
(horto005@maroon.tc.umn.edu) has done with a little ingenuity and a few scraps
lying around the attic.

Not being dissuaded by lack of a few screws, Dr Horton has managed to build a
working shaker unit for less than US$30 and a few hours investment.  His
shaking incubator was built from a cardboard box, a record player, a light
bulb, a thermostat, tape, string and some other junk found lying around.  A
swinging platform is suspended from strings from the top of the box and
positioned just above the rotating record player. A bolt attached to the
turntable pulls the platform around with it as it oscillates.

Granted the cardboard box is not the prettiest piece of equipment in the lab,
but it can get the job done.  He has written that his box contraption is fully
functional and he has even used it to grow bacterial cultures for the cloning
of a cDNA sequence for a novel human neurotransmitter receptor subunit.  He
wrote that not only can record players be made into incubators, but they can
also be converted into rotating water baths or platform orbital shakers to wash
gels and membranes or shake test tubes for much less than you would spend on a
new one from a scientific supply company. In addition, the homemade version has
three speeds of 33, 45, and 78 r.p.m. and can be rigged with a working
hip-wobbling cardboard cut-out of Elvis to keep the lab rats entertained during
those long wash cycles.

If you are interested in more details or obtaining the blueprints, the complete
directions for making Dr Horton's homemade shaking incubator can be found at
http://biosci.cbs.umn.edu/misc/horton/shaker.html or refer to the bionet
archives for the posted version. Unfortunately, you might have to unpack the
archived version as there are embedded images. However, by using the mpack
program available from ftp://ftp.andrew.cmu.edu/pub/mpack you can decode them.
For details on how to do this, see his article on sending images by Email in
the upcoming May 1996 issue of BioTechniques.

What goes around, comes around
******************************
In a past Methods and reagents column, I wrote about a new and interesting
method of amplifying DNA by using a boomerang technique (see TiBS 1995 20,
372-373).  Unlike PCR, Boomerang DNA Amplification (BDA) allows one to amplify
DNA sequences using only a single primer.  This past month Kevin Ahern
(ahernk@ava.bcc.orst.edu) opened the BDA Web Page at
http://www.orst.edu/~ahernk/bdapatent.html, which provides many more details
about the BDA process, the history of the invention and the patent awarded to
BDA (US Pat. No. 5 470 724).


References
**********

[1] Kumar, T. K. S. et al. (1993)
    Anal. Biochem. 213, 226-228

[2] Beis, A. and Lazou, A. (1990)
    Anal. Biochem. 190, 57-59

[3] Gray, W. R.(1993)
    Protein Sci 2, 1732-1748

[4] Singh, R. (1994)
    BioTechniques 17, 263-265

[5] Tasheva, B. and Dessev, G. (1983)
    Anal. Biochem. 129, 98-102

[6] Ochs, D. (1983)
    Anal. Biochem. 135, 470-474

[7] Kruchinina, N. G. and Gresshoff, P. M. (1994)
    BioTechniques 17, 280-282

[8] Shapiro, S. Z. (1987)
    J. Immun. Methods 102, 143-146

[9] Riches, P. G., Polce, B. and Hong, R. (1988)
    Anal. Biochem. 110, 117-121

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Any statements made by the author are not meant to advocate the use of a
particular commercial product or endorse any company. All opinions are
those of the author and do not reflect the opinion of the National Cancer
Institute or the National Institutes of Health.

Copyright: This manuscript is not copyrighted by Elsevier Publishing Company.
However, you may not reproduce any portion for resale or edit the text for
redistribution, sale, or otherwise without written permission from the author.

You found this at the World Wide Web (WWW) Uniform Resource Locator (URL)
ftp://ftp.ncifcrf.gov/pub/methods/TIBS/may96.txt

Any reference to this column must be cited as the following published article:
Hengen, P. N. 1996. Methods and reagents - Eliminating banding artifacts
from SDS-PAGE. Trends in Biochemical Sciences 21(5):191-193.

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* Paul N. Hengen, Ph.D.                           /--------------------------/*
* National Cancer Institute                       |Internet: pnh@ncifcrf.gov |*
* Laboratory of Mathematical Biology              |   Phone: (301) 846-5581  |*
* Frederick Cancer Research and Development Center|     FAX: (301) 846-5598  |*
* Frederick, Maryland 21702-1201 USA              /--------------------------/*
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