ANALYTICAL
`
`BIOCHEMISTRY
`
`67, 317-35’
`
`(1975)
`
`of Submicrogram
`Quantitation
`Using Coomassie
`Brilliant
`Dodecyl
`Sulfate-Polyacrylamide
`
`of Protein
`Amounts
`Blue R on
`Slab-Gels
`
`Sodium
`
`slab-gel system was used to
`A sodium dodecyl sulfate (SDS)-polyacrylamide
`study the use of Coomassie brilliant blue (CB) as a quantitative stain. Quantita-
`tion curves are shown
`for tubulin, cytochrome c, histone, and actin from gels
`stained with CB. A comparison was made of three identical gels using an actin
`sample and stained with CB, fast green and buffalo black. CB staining was found
`to be quantitative
`in the 0.05-2.75pg
`range as well as possessing an order of
`magnitude
`increase in sensitivity over other stains tested.
`
`elec-
`polyacrylamide-gel
`in quantitative
`the recent work
`Much of
`(PAGE)
`is concerned with
`the stains and staining proce-
`trophoresis
`dures. Some of the different stains used are fast green (l), amido black
`(2), and Coomassie brilliant
`blue (CB) (3). In most cases only a small
`percentage of the dye ever binds to protein. This prompted Datyner and
`Finnimore
`(4) to develop a method
`in which stain and protein are mixed
`under conditions
`favorable
`to
`the formation
`of covalent bonds before
`electrophoresis.
`They report a sensitivity down to 0.2 pg with some pro-
`teins. Another method, employed by Watkin and Miller
`(5), attempts
`to
`avoid
`the problems
`inherent
`in the use of dyes and staining by scanning
`gels immediately
`after electrophoresis
`under uv light at 280 nm. How-
`ever, as is aptly pointed out (5), the problem of variable affinity
`for stain
`is exchanged
`for one of different
`relative absorbancies of light at 280 nm.
`While CB is held by most workers
`to be the most sensitive dye avail-
`able,
`there has been some doubt as to its value as a quantitative
`dye.
`Fishbein
`(3) has shown
`that CB could be quantitated
`in the
`l-22-pg
`range. The major complaint
`against
`the use of CB as a quantitatively
`staining dye is that it shows deviation
`from Beer’s Law at lower protein
`concentrations
`than the other dyes.
`to various proteins
`In this study CB was shown to bind quantitatively
`at submicrogram
`concentrations
`not previously
`reported. The
`impor-
`tance of the appropriate
`slab-gel system
`is also emphasized.
`
`MATERIALS AND METHODS
`Gels. The polyacrylamide
`thin (16 X 12 X 0.08 cm) slab-gel system of
`Laemmli
`(6) and O’Farrell
`et al. (7) was used, giving gels with an
`acrylamide
`concentration
`of 12% and a sodium dodecyl sulfate
`(SDS)
`
`Copyright @ 1975 by Academic
`All
`rights of reproduction
`in any
`
`Press.
`form
`
`Inc.
`reserved.
`
`347
`
`GNE 2006
`Page 1
`
`

`
`348
`
`SHORT
`
`COMMUNICATIONS
`
`than 72 nor less
`the spacer gel and
`
`of 0.1%. Gels were made up no more
`concentration
`than 12 hr before use and were run at 10 mA
`through
`15 mA once into
`the lower gel.
`lot
`Chemical,
`(Sigma
`blue R
`Staining.
`Coomassie
`brilliant
`and
`#23C-0950),
`fast green FCF
`(Sigma Chemical,
`lot #7OC-0580),
`buffalo black NBR
`(Allied Chemical,
`lot #7OC-0580) were used at a
`concentration
`of 0.1%
`in 25%
`trichloroacetic
`acid (TCA). Destaining
`took 2-3 days employing
`changes of 8% acetic acid. Gels were consid-
`ered completely
`destained when there was no visible background
`stain
`remaining
`in the gel.
`10%
`Samples. All proteins were made up in a sample buffer containing
`glycerol, 5% 2-mercaptoethanol,
`2% SDS, 8 M urea, 0.0625 M Tris, pH
`6.8 with HCl.
`Histone
`from
`calf
`thymus
`(Sigma Chemical,
`lot
`#6OC-2650) was prepared as a 1 mg/ml
`solution
`in sample buffer. Other
`samples
`included
`cytochrome
`c (horse heart, Sigma Chemical,
`lot
`#6OC-2650) made up to 0.02 mg/ml,
`tubulin purified by vinblastine
`pre-
`cipitation
`at a concentration
`of 0.012 mg/ml, and rabbit muscle actin,
`diluted
`to a final concentration
`of 0.025 mg/ml.
`Quantitation.
`The stained bands were cut out of the slab gels using a
`razor blade and placed along
`the inside wall of a Gilford 24 12 rectangu-
`lar cuvette
`to which
`they adhered. Fresh,
`filtered 8% acetic acid was
`used to fill
`the cuvette
`in all work reported. The gels were scanned at
`590 nm (red) with
`the use of a Gilford 240 spectrophotometer
`with a
`grid slit plate 2.36 x 0.05 mm and a slit width of 0.36 mm. Graphs were
`made by using a Gilson
`linear
`transport apparatus
`to move
`the gels past
`the spectrophotometer
`beam at the slowest scan speed (0.5 cmlmin) and
`recorded on a Texas
`Instruments
`chart
`recorder at a speed of 7.6
`cmlmin.
`of 13 samples. No attempt was
`Each gel had spaces for a maximum
`made to compare
`results from different gels. Four readings were taken of
`each gel as follows: The gel was read, flipped over and read again, a thin
`slice was taken off lengthwise and the gel was read again, and a second
`lengthwise slice was taken off for the fourth reading. This gave four dis-
`tinct points on the protein band which were read and quantitated.
`The
`number of readings was later reduced
`to two per sample. The means of
`the areas under
`the peaks, determined
`by weighing on a Mettler balance,
`of the readings
`for each sample were then taken as a quantitative
`value.
`
`RESULTS AND DISCUSSION
`tissue homogenates
`of either pure proteins or whole
`Electrophoresis
`gels proved
`to be of insufficient
`sensitivity,
`particularly
`using cylindrical
`when compared with that of the slab-gel system. Three
`to five times
`the
`amount of protein
`is required when using a cylindrical
`(7-mm diameter)
`
`GNE 2006
`Page 2
`
`

`
`SHORT
`
`COMMUNICATIONS
`
`349
`
`.2
`
`.
`
`5
`
`2s
`z
`0
`
`.40-
`
`.35-
`
`.30-
`
`.25-
`
`.20-
`
`.15-
`
`.lO-
`
`OS-
`
`r
`
`I
`I
`I
`5
`10
`15
`Sample
`Volume
`(k.l)
`of four different proteins (tubulin. 0.012 pg/wl; actin, 0.025 /.~g//.~l;
`1. Quantitation
`FIG.
`cytochrome c, 0.02 pg/pl, and histone) at four sample volumes. Means and ranges are
`shown for each point.
`
`I
`20
`
`the greatest
`gel due to the greater volume of the gel matrix. However,
`problem
`in using cylindrical
`gels was found
`to be in the staining.
`It was
`found
`that the stain does not penetrate
`the cylindrical
`gels uniformly
`or
`completely
`and thus could not be used for quantitative
`purposes. Pene-
`tration of the dye throughout
`the gel can be checked by slicing
`the gel
`with a razor blade
`in the middle of a peak and looking
`to see if the stain
`has penetrated
`to the center. When
`the penetration
`is incomplete
`there is
`a ring of stain with a clear center. Staining procedures were varied
`in ef-
`forts to get complete
`staining, but
`this was never achieved with cylin-
`drical gels even after 2 days of staining or with
`the use of acetic acid,
`methanol,
`or trichloroacetic
`acid solutions. However,
`it was discovered
`that staining
`for 30 min.
`in a 0.1% CB solution
`in 25% TCA was suf-
`ficient
`for complete,
`uniform
`staining of a slab gel. When destained
`in
`7-10%
`acetic acid,
`there
`is no spreading of bands and only gradual
`fading of intensity of bands due to leaching of the dye.
`
`GNE 2006
`Page 3
`
`

`
`350
`
`SHORT COMMUNICATIONS
`
`.9 0
`
`.60
`
`.70
`
`.60
`
`.50
`
`.40
`
`30
`
`.20
`
`.lO
`
`M
`
`.'
`. 5
`
`AZ
`
`:
`n.
`
`0
`
`5
`0:o
`
`I
`.25
`
`I
`.50
`
`I
`.7 5
`
`8
`1.0
`
`I
`1.25
`
`g Actin
`u
`In each
`FIG. 3. Ten actin samples were made up with different protein concentrations.
`case the same sample volume (10 ~1) is used to give concentrations
`from 0.05-1.X
`pg.
`This graph represents one gel.
`
`in amino acid
`A variety of proteins were chosen to reflect differences
`composition,
`net charge, and molecular weight. The data are shown (Fig.
`1) for a histone protein
`(R,, 0.591),
`tubulin, actin, and cytochrome
`c. It is
`noted
`that in most of these graphs at the greatest sample volume
`(20 ~1)
`the value
`is slightly
`higher
`than
`that predicted
`from
`the
`line drawn
`through
`the other points. This was thought
`to be due to the increase
`in
`sample volume
`rather
`than a real deviation
`resulting
`from staining. This
`is supported
`by results shown
`in Fig. 2 in which
`ten different actin
`samples were prepared
`in different concentrations
`so that, when put on
`the gel, the same sample volume of 10 ~1 was used but different amounts
`of protein were in each sample
`(0.25-1.25
`pg).
`
`GNE 2006
`Page 4
`
`

`
`3.51
`
`SHORT
`
`COMMUNICATIONS
`
`040
`
`Black
`Green
`. Fast
`0 Coommassie
`
`I o Buffalo
`
`Blue
`
`Volume
`(~4 L)
`FIG. 3. Results of quantitation of three actin gels, identical
`the stain employed.
`
`in every respect. except fol
`
`fast green and buffalo
`of CB,
`the sensitivities
`Figure 3 illustrates
`the values from
`the CB-stained
`black. At the same protein concentration
`greater
`than peaks
`from gels
`gel are almost an order of magnitude
`stained with either
`fast green or buffalo black. The
`lowest amount of
`protein quantitated was 0.05 pg and could easily be seen by the eye. The
`greatest amount quantitated was 2.75 pg per band. We were thus able to
`quantitate protein bands with almost a hundred-fold
`range of sensitivity.
`Fishbein
`reports a range of I-55 pg which probably
`reflects differences
`in the sensitivity of the spectrophotometer
`used in quantitating.
`It should
`be noted
`that with different
`instrumentation
`it should be possible
`to shift
`the range of concentrations which are quantitatable with the use of CB.
`The utility of such a technique
`is perhaps best found
`in cellular and
`molecular
`research rather
`than as a strictly analytical method such as the
`Lowry protein determination.
`The ability of PAGE
`to separate proteins
`
`GNE 2006
`Page 5
`
`

`
`352
`
`SHORT COMMUNICATIONS
`
`them on a submicrogram
`and quantitate
`any other existing methodology.
`
`scale cannot be approached by
`
`REFERENCES
`1. Gorovsky, M. A., Carlson. K., and Rosenbaum, J. L. (1970) Anal. Biochem. 35,
`359-370.
`2. Kruski, A. W., and Narayan, K. A. (1968) Biochim. Biophys. Acta 168, 570-572.
`3. Fishbein, W. N. (1972) Anal. Biochem. 46, 388-401.
`4. Datyner, A., and Finnimore, E. D. (1973) Anal. Biochem. 55, 479-491.
`5. Watkin, J. E., and Miller, R. A. (1970) Anal. Biochem. 34, 424-435.
`6. Laemmli, U. K. (1970) Nature
`(London) 227, 680-685.
`7. O’FarrelI, P. Z., Gold, L. M.. and Huang, W. M.,
`5499-5501.
`
`(1973) J. Biol. Chem. 248,
`
`KAHN
`RICHARD
`ROBERT W. RUBIN
`
`Division of Natural Sciences
`New College
`Sarasota, Florida 33578
`Received August 26, 1974; accepted March 3. 1975
`
`GNE 2006
`Page 6