Report Fernanda Balem
10/14/05
I – Introduction
The commonly used stains, Coomassie brilliant blue and the more sensitive silver
nitrate allow for detection of most proteins separated by SDS-PAGE. However, there are
several classes of highly negatively charged proteins that are detected poorly by these
typical stains. This is because the negatively charge of Coomassie blue and silver nitrate
are repelled by these highly acidic proteins. Therefore, other dyes, specifically those of a
cationic nature, have been used to stain these proteins on polyacrylamide gels (Harvey A.
Goldeberg and Kevin J. Warner, 1997). The metachromatic cationic carbocyanine dye
“Stains-all” (1-ethyl-2-{3-(1-ethyl-naphthol[1,2-d]thiazoline-2-ylidine)-2-methylpropenyl} (Fig
1) is one of the dyes that can bind to highly acidic proteins .It can also be used to distinguish
calcium-binding proteins (CaBP) from others. CaBP are stained blue or purple by Stainsall while others proteins are stained red or pink (Sharma and Balasubramanian, 1991).
Fig. 1. Structural formula of the dye Stains-all (taken from http://omlc.ogi.edu/spectra/PhotochemCAD/html/stainsall.html on
10/11/05)
The colors of the proteins bands observed on polyacrylamide gels are due to five
different types of complex states. Their corresponding approximate spectral band maxima
have been designated as: α, β, γ, J, βα and S. The α band has an absorption maximum at
570 nm, the β band at 535 nm, the γ band at 500-510 nm, the J band at 600-650 nm, a βα
is a hybrid or mixture state with absorption maxima at 550 nm, and the S band at 470 nm
(Sharma and Balasubramanian, 1991). Each state depends on the adsorption of the dye by
the macromolecule and on the nature and conformation of the macromolecule. Blue
staining (J state, 610-650 nm) results from the interaction of individual dye molecules at
anionic sites such as sialic acid or phosphoryl groups in the protein
Densitometric scans of Stains-all-stained gels revealed that interaction of the dye
with Ca2+-binding proteins changed the absorption spectrum of the dye. The dye-protein
complex absorbed maximally at 615 nm, reflecting a state that results from the binding of
individual dye molecules at anionic sites. Stains-all also interacted with undenatured
Ca2+- binding proteins in aqueous solution forming a complex absorbing maximally at
600 nm. These results suggest that the interaction of he dye with anionic sites within
these Ca2+-binding proteins produces the dye-protein complex which absorbs at 600-615
nm (Campbell et al., 1983).
Campbel et al, show that several well-known high or low affinity Ca2+- binding
proteins could be easily identified by their blue staining in the presence of Stains-all.
1
Report Fernanda Balem
10/14/05
These findings are consistent with the interpretation that Stains-all binds to anionic sites
within Ca2+-binding proteins and the resulting J complex gives raise to the blue-staining
property. Previous studies have shown that Stains-all stains sialoglycoproteins (i.e.
glycophorin) and phosphoproteins (i.e. phosvitin and casein) blue while staining most
proteins red or pink. The blue staining of these proteins resulted from the complex
formed between the dye and the anionic sites created by silica acid residues or
phosphoryl groups in these proteins. Although observations support the view that Ca+binding proteins will stain blue with Stains-all, it is probable that the extent of blue
staining is dependent on the number of Ca2+-binding sites in the protein.
It is proved that the stains all Dye binds to the anionic site of the protein and
shows J band. This J band could be used to explore the conformation status of the protein.
The interaction of the cationic carbocyanine dye Stains-all with the eye lens proteins
crystallins has been studied (Sharma et al. 1989) and it suggests that analysis of the
metachromasia induced in the dye by these and other proteins is responsive to the
conformational status of the region to which it binds in a protein.
The interaction of Calcium-binding proteins (CaBP) with Stains-all could be
specific and unspecific. Polyglutamic acid binds unspecifically, high capacity and low
affinity. Whereas Calmodulin binding to Stains-all is specific, high affinity and is derived
by few binding sites. To investigate the specific and unspecific interaction of Stains-all
we explored the spectral changes imparted by these interactions. BSA and Calmodulin
are the control experiment to further our studies to find the conformational states of Garp2. Garp-2 acts as Ca buffer and it shows multiple bands on Stains all gel ranging color
from blue to pink. We hypothesize that these multiple bands are different conformations
of Garp-2 protein, which could be further investigated by analysis of the metachromasia
induced in the dye by these Garp-2 proteins. These studies could also open doors to
understand the environment of the acidic residues in Ca binding proteins, in spite of the
known and suggested vital role of the clusters of acidic residues in Ca2+ -binding
proteins.
II – Materials and Methods
The protocol used for preparing the dye solutions and the protein-dye complexes
was the same described by Sharma et al., 1989. The Stains-all solutions were prepared
dissolving the dye in ethylene glycol to about 0.28mM and the actual concentration of the
dye determined based on a molar extinction coefficient value at 578 nm of 1.13 x 10 5 in
ethylene glycol. In order to make the complexes, Bovine serum albumin (BSA),
Polyglutamic acid and Calmodulin were dissolved in 2mm MOPS buffer, pH 7.2,
containing 30% ethylene glycol, to which an aliquot of the stock solution of the dye was
added, and the mixture kept in the nutator for one hour in the dark. The visible spectra
was recorded from 400nm to 700nm using perkin elmer spectrophotometer. 1mM CaCl2
was added to the mixture of protein and Stains all and incubated for 30 min in dark. The
spectra were again recorded to check the effect of Ca ions on the interaction of Stains all
with the respective protein samples.
2
Report Fernanda Balem
10/14/05
III – Results and Discussion
The stability of the Stains all stock solution was checked by recording the spectra
for 5 days. It seems that Stains all is stable in ethylene glycol, but it dissolves more and
more each day. The best way would be to dissolve the stains all and then centrifuge it to
get rid of the not dissolved dye particles to get a stable concentrated ready stock of Stains
all in Ethylene glycol.
Stability of Stains All
1.4
1.2
Absorbance
1
Day 0
0.8
Day 1
Day 2
0.6
Day 3
0.4
Day 4
0.2
673
642
612
581
550
520
489
459
428
400
0
-0.2
Fig. 1: Stability of Stains all
W a ve le ngth
Further we repeated the experiment of stains all with varying levels of ethylene
Glycol. Visible spectrum of Stains-all (7.9μM) in 2 mM Mops, pH 7.2 at different
ethylene glycol levels. A, 3%; B, 10%; C, 30%; D, 60%; and E, 98% were recorded.
S ta in s a ll w ith E th y le n e G ly c o l
1
Absorbance
0.8
3%
0.6
10%
30%
0.4
60%
98%
0.2
673
642
612
581
550
520
489
459
428
7.9M in E th.G ly c ol
400
0
-0.2
W a ve le n g th
Fig. 2: Visible spectrum of stains all at different Ethylene Glycol levels .
3
Report Fernanda Balem
10/14/05
In the absence of protein, Stains-all (7.9 μM) in 2 mM Mops, pH 7.2, 23 °C, has a
visible absorption spectrum which is dominated by a band of the γ-type at 510 nm, with
shoulders at 530 and 570 nm (Fig. 2). In the presence of 30% ethylene glycol, only two
prominent bands are present, at 535 and 575 nm. The 575 nm α-band, which has been
observed for the dye in organic solvents, probably corresponds to monomer. The 535 nm
β-band appears to arise from a lower associated species, which is nonsedimentable,
perhaps from dimer. There is little or no indication of any J-band at 650 nm, which, in the
case of free dye, arises from extensively aggregated species. Also, bands of the γ (500510 nm), βα (550 nm), and S (470 nm) types, which occur in aqueous solutions of free
dye, are not observed in 30% ethylene glycol. Further addition of ethylene glycol results
in progressive loss of the β-band and the predominance of the monomeric α –band
(Caday and Steiner, 1985).
To check the interaction of Stains all with the Calcium binding proteins, we
repeated the experiments of interaction of PGA, BSA and Calmodulin with stains all.
Interaction of Polyglutamic acid (PGA) with Stains-all
BL
673
642
612
-1
581
5
6
550
0
520
3
4
489
1
459
1
2
428
2
400
Absorbance
PGA
7
8
Wavelength
Fig. 3: Visible spectrum of PGA with Stains all complexes with 2mM MOPS,30% ethylene
glycol, pH 7.2. The dye-PGA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1
respectively.
1
673
642
612
581
550
520
489
459
2
428
1
0.5
0
-0.5
-1
-1.5
-2
400
Absorbance
PGA difference spectra
3
4
5
6
Wavelength
7
Fig. 4: Shows the difference spectra from Stains all/PGA.
4
Report Fernanda Balem
10/14/05
1
0.5
530 nm
0
-0.5
50
25
12.5
6.25
3.12
1.56
557 nm
1
581 nm
-1
630 nm
-1.5
-2
Fig. 5: Prominent peaks of difference spectra.
Interaction of Polyglutamic acid + CaCl2 with Stains-all
BL
PGA + CaCl2
1.5
2
1
3
0.5
4
5
673
642
612
581
550
520
489
459
-0.5
428
0
400
Absorbance
1
6
7
Wavelength
8
Fig:6 : visible spectrum of PGA + CaCl2 with Stains all complexes with 2mM MOPS,30% ethylene glycol,
pH 7.2. The dye-PGA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
673
642
612
581
550
-0.5
520
3
489
2
0
459
1
0.5
428
1
400
Absorbance
PGA + CaCl2 difference spectra
6
-1
5
-1.5
6
Wave le ngth
7
Fig 7 Shows the difference spectra from Stains all/PGA + CaCl2.
5
Report Fernanda Balem
10/14/05
1
0.5
530 nm
0
25
50
-0.5
557 nm
1
1.56
3.12
6.25
12.5
581 nm
-1
630 nm
-1.5
-2
Fig 8: Prominent peaks of difference spectra.
Interaction of Calmodulin with Stains-all
Calmodulin 3x Diluted
Base Line
1
1
2
0.5
3
4
5
673
642
612
581
550
520
489
459
-0.5
428
0
400
Absorbance
1.5
6
7
Wavelength
Fig 9: visible spectrum of Calmodulin with Stains all complexes with 2mM MOPS,30% ethylene glycol,pH
7.2. The dye-Calmodulin mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
Calmodulin3x diluted - difference spectra
1
2
3
673
642
612
581
550
520
489
459
-0.5
428
0
400
Absorbance
1
0.5
4
5
6
-1
Wavelength
Fig 10: Shows the difference spectra from Stains all/Calmodulin
6
Report Fernanda Balem
10/14/05
Calmodulin Peaks
1
634nm
0.5
570nm
0
25
50
-0.5
12.5
530nm
1
3.12
6.25
490nm
-1
Fig 11: Prominent peaks of difference spectra.
Interaction of Calmodulin + CaCl2 with Stains-all
1.2
BL
1
0.8
1
0.6
0.4
3
2
4
0.2
0
673
642
612
581
550
520
489
459
5
428
-0.2
400
Absorbance
Calmodulin 3x diluted + CaCl2
6
7
Wavelength
Fig 12: Visible spectrum of Calmodulin + CaCl2 with Stains all complexes with 2mM MOPS,30%
ethylene glycol, pH 7.2. The dye-CAlmodulin mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1
respectively.
Calm3x diluted+CaCl2-difference spectra
673
642
612
581
550
520
3
-0.2
489
2
0
459
1
0.2
428
0.4
400
Absorbance
0.6
4
-0.4
5
-0.6
6
-0.8
Wavelength
Fig 13: Shows the difference spectra from Stains all/Calmodulin + CaCl2.
7
Report Fernanda Balem
10/14/05
Calm +CaCl2 Peaks
0.5
648nm
576nm
0
50
25
12.5
6.25
3.12
554nm
1
-0.5
533nm
493nm
-1
Fig 14: Prominent peaks of difference spectra.
Interaction of BSA with Stains-all in ~1 hour – Experiment 1
BSA
BL
2.5
1
Absorbance
2
2
1.5
3
1
4
0.5
5
673
642
612
581
550
520
489
459
428
-0.5
400
0
Wavelegth
6
7
8
Fig 15: Visible spectrum of BSA with Stains all complexes with 2mM MOPS, 30% ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
BSA/difference spectra
1
1
2
673
642
612
581
550
520
-0.5
489
4
459
3
0
428
0.5
400
Absorbance
1.5
5
6
-1
Wavelength
7
Fig 16: Shows the difference spectra from Stains all/BSA
8
Report Fernanda Balem
10/14/05
BSA Peaks
1.2
1
0.8
0.6
579nm
0.4
519nm
0.2
442nm
0
-0.2
50
12.5
25
1
1.56
3.12
6.25
-0.4
-0.6
Fig 17: Prominent peaks of difference spectra.
Interaction of BSA + CaCl2 with Stains-all
BSA + CaCl2
Absorbance
2
BL
1.5
1
1
2
3
0.5
4
0
673
642
612
581
550
520
489
459
428
400
-0.5
5
6
7
Wavelength
Fig 18: Visible spectrum of BSA + CaCl2 with Stains all complexes with 2mM MOPS, 30% ethylene
glycol, pH 7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
BSA+CaCl2- Difference spectra
1.5
1
1
2
3
0.5
4
673
642
612
581
550
520
489
459
-0.5
428
0
400
Absorbance
2
5
6
-1
Wavelength
Fig 19: Shows the difference spectra from Stains all/BSA
9
Report Fernanda Balem
10/14/05
BS A + CaCl2
2
1. 5
1
576nm
0. 5
534nm
0
451nm
-0. 5
-1
Fig 20: Prominent peaks of difference spectra.
Interaction of BSA with Stains-all in ~ 1 hour – Experiment 2
BL
1
2
3
4
674
643
613
582
551
521
490
460
5
429
0.14
0.12
0.1
0.08
0.06
0.04
0.02
0
-0.02
-0.04
401
Absorbance
2 exp with BSA -
6
7
8
Wavelength
Fig 21: Visible spectrum of BSA with Stains all complexes with 2mM MOPS, 30% ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
1
674
643
613
582
551
521
490
460
2
429
0.03
0.02
0.01
0
-0.01
-0.02
-0.03
-0.04
-0.05
-0.06
401
Absorbance
Difference (SA/SA+BSA) 2 Exp
3
4
5
6
7
Wavelength
Fig 22: Shows the difference spectra from Stains all/BSA
10
Report Fernanda Balem
10/14/05
0.04
652nm
0.02
572nm
0
50
-0.02
25
12.5
6.25
3.12
1.56
555nm
1
535nm
-0.04
493nm
-0.06
Fig 23: Prominent peaks of difference spectra.
Interaction of BSA with Stains-all (after ~ 24 hours)
BSA 2Exp part B
Absorbance
0.1
BL
0.08
1
0.06
2
3
0.04
4
0.02
5
0
674
643
613
582
551
521
490
460
429
6
401
-0.02
7
8
Wavelength
Fig 24: Visible spectrum of BSA with Stains all complexes with 2mM MOPS, 30% ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
BSA -difference spectra - part B
0.1
1
2
0.06
3
0.04
4
0.02
5
674
643
613
582
551
521
490
460
-0.02
429
0
401
Absorbance
0.08
6
7
-0.04
Wavelength
Fig 25: Shows the difference spectra from Stains all/BSA
11
Report Fernanda Balem
10/14/05
BSA Exp2-after 24 hours
0.1
0.08
0.06
654nm
576nm
0.04
557nm
0.02
537nm
0
-0.02
50
25
12.5
6.25
3.12
1.56
502nm
1
-0.04
Fig 26: Prominent peaks of difference spectra.
Interaction of BSA with Stains-all (after ~ 48 hours)
BSA Exp 2 part C
Absorbance
0.1
Bl
0.08
1
0.06
2
3
0.04
4
0.02
5
0
673
642
612
581
550
520
489
459
428
400
-0.02
6
7
8
Wavelength
Fig 27: Visible spectrum of BSA with Stains all complexes with 2mM MOPS, 30% ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
BSA - difference spectra - part C
0.08
2
0.04
3
4
674
643
613
582
551
-0.02
521
6
490
0
460
5
429
0.02
401
Absorbance
1
0.06
7
Wavelength
Fig 28: Shows the difference spectra from Stains all/BSA
12
Report Fernanda Balem
10/14/05
BSA (48 hours)
0.08
0.07
0.06
574nm
0.05
555nm
0.04
537nm
0.03
441nm
0.02
0.01
0
50
25
12.5
6.25
3.12
1.56
1
Fig 29: Prominent peaks of difference spectra.
Interaction of BSA witn Stains-all in ~1 hour – Experiment 3
BSA 3Exp
0.2
BL
1
Absorbance
0.15
2
0.1
3
4
0.05
5
673
642
612
581
550
520
489
459
428
-0.05
400
0
6
7
8
Wavelength
Fig 30: Visible spectrum of BSA with Stains all complexes with 2mM MOPS, 30% ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
Difference (SA/SA+BSA) 3 Exp
0.03
1
0.02
2
3
0.01
4
-0.02
674
643
613
582
551
521
490
460
-0.01
429
0
401
Absorbance
0.04
5
6
7
-0.03
Wavelength
Fig 31: Shows the difference spectra from Stains all/BSA
13
Report Fernanda Balem
10/14/05
0.04
0.03
0.02
576nm
0.01
558nm
535nm
0
50
25
12.5
6.25
3.12
1.56
497nm
1
-0.01
-0.02
-0.03
Fig 32: Prominent peaks of difference spectra.
Interaction of BSA with Stains-all (after ~ 24ours)
BSA exp3 part B
5
0.01
6
0
7
-0.01
673
0.02
642
4
612
0.03
581
3
550
0.04
520
2
489
0.05
459
1
428
0.06
400
Absorbance
0.07
Wavelength
Fig 33: Visible spectrum of BSA with Stains all complexes with 2mM MOPS, 30% ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
BSA Exp 3 - after 24 hours- Difference spectra
0.06
1
0.04
2
3
0.02
4
673
642
612
581
550
520
489
459
-0.02
428
0
400
Absorbance
0.08
5
6
-0.04
Wavelength
Fig 34: Shows the difference spectra from Stains all/BSA
14
Report Fernanda Balem
10/14/05
BSA 3 Exp -part B
0.07
0.06
0.05
0.04
0.03
0.02
0.01
0
655nm
572nm
538nm
-0.01
-0.02
-0.03
50
25
12.5
6.25
3.12
1
Fig 35: Prominent peaks of difference spectra.
Interaction of BSA with Stains-all in ~ 1hour – Experiment 4
BL
BSA 4Exp
674
-0.05
643
5
613
0
582
4
551
0.05
521
3
490
0.1
460
2
429
0.15
401
Absorbance
1
6
7
Wavelength
8
Fig 36: Visible spectrum of BSA with Stains all complexes with 2mM MOPS, 30% ethylene glycol, pH
7.2. The dye-BSA mole ratios are Control, 50, 25, 12.5, 6.25, 3.12, 1.56 and 1 respectively.
673
642
612
581
550
-0.02
520
2
489
0
459
1
428
0.02
400
Absorbance
BSA 4Exp difference spectra
3
4
-0.04
5
-0.06
6
Wavelength
7
Fig 37: Shows the difference spectra from Stains all/BSA
15
Report Fernanda Balem
10/14/05
BSA Exp 4
0.01
0
50
25
12.5
6.25
-0.01
-0.02
-0.03
3.12
1.56
1
575nm
553nm
534nm
500nm
-0.04
-0.05
Fig 38: Prominent peaks of difference spectra.
IV Future Plans
We plan to investigate about the interaction of Garp-2 with stains all dye to help
us understand if this dye could be used as a system to find different conformation of the
Garp-2 protein.
We are trying to find optimum buffer conditions to concentrate Garp-2 for NMR
studies.
V References
1. Caday, C.G., and Steiner, R.F. (1985) J. Biol. Chem. 260, 5985-5990.
2. Sharma, Y., and Balasubramanian, D. (1991) title, In CW Heizmann, ed. Novel
Calcium-Binding Proteins. Speinger- Verlaz, Berlin, pp 51-61.
3. Campbell, K.P., MacLennan,D.H. and Joegensen, A.O. (1983) J. Biol. Chem.258,
11267-11273.
4. Goldberg, H.A., and Warner, K.J. (1997) Analytical Biochemistry 251, 227-233.
5. Sharma, Y., Rao,R.M., Rao, S.C., Krishna, a.G., Somasundaram, T.,
Balasubramanian, D. (1989) J. Biol. Chem. 264, 20923-20927.
16