new fluorescence dyes for protein gel stains
DESCRIPTION
The poster, New Fluorescence Dyes For Protein Gel Stains, presented by Sigma-Aldrich.TRANSCRIPT
SummaryThree new fl uorescent protein gel stains, LUCY-506, LUCY-565 and LUCY-569 have been developed. The profi le of each of the dyes has been established: LUCY-506 shows highest sensititivity. LUCY-565 allows neutral gel-staining (e.g. before Western blotting) and LUCY-569 excels by a linear response over an extraordinary broad linear dynamic range.
Motives• Standard procedures for on gel protein staining either lack sensitivity for the lower ng/band
protein range (e.g. Coomassie Blue stains), are labour-intensive (silver stains), or show poor MS-compatibility.
• Commercially available fl uorescent stains overcome those disadvantages only partially and are generally expensive.
• Conventional stains are not designed for state-of-the art detectors such as CCD camera imagers and laser scanners and generally show limited linearity in signal intensity vs. protein/band relation which make them unsuitable for protein quantifi cation on gel.
Staining ProtocolsStandard procedure
Prestainingprocedure
Stain afterfi xation
NativePAGE stain
Neutral stainbefore WB
post-electrophoreticstain in HOAc;time required: 1h
dye in cathode-running-buffer with subsequentdestaining inHOAc; timerequired:15-60 min
TCA-fi xation; SDS-rinse;stain in NaOAc; time required: 1h 45 min
run gel SDS-free;rinse gel in SDS;then standard procedure;time required:1h 30min
postelectrophoreticstain in water; timerequired: 1h;continue withWestern-Blot transfer
LUCY-506 +++ ++ + +++ -
LUCY-565 + + ++ +++ +++
LUCY-569 +++ ++ ++ +++ -
Table 1. Suitability and performance of LUCY stains for various staining procedures.
Detection Workfl ow
Protein Mix
Gel Electrophoresis
LUCY-506LUCY-565LUCY-569
Gel-Stain
Imaging
LUCY-565
Band/Spot Excision Digest
MS
Western Blot
Membrane
Antibody Decoration
Optimum Detection Settings
LUCY-506 LUCY-565 LUCY-569ImagingDevice
Illumination Filter Sensitivity Filter Sensitivity Filter Sensitivity
Polaroid Camera
Dark Reader®
(lmax. ~450 nm)
orangefi lter
+ not tested not tested
CCDCamera
UV screen
(lmax. ~310 nm)
535 nm band pass
++ not tested not tested
590 nm band pass
- 590 nm band pass
++ 590 nm band pass
+++
Dark Reader®
(lmax. ~450 nm)
590 nm band pass
+++ 590 nm band pass
+ 590 nm band pass
+
amberfi lter
+ amber fi lter
- amber fi lter
-
LaserScanner
473 nm laser 520 nm cut off
+++ 520 nm cut off
- 520 nm cut off
-
532 nm laser 580 nm cut off
- 580 nm cut off
+++ 580 nm cut off
++
Table 2. Imaging devices, methods and performance.
New Fluorescence Dyes For Protein Gel StainsBernhard Schönenberger1, Pierre Nording1, Sergiy Yarmoluk2, Alex Rück1, Monika Bäumle1, Michael Weber1
1Sigma-Aldrich GmbH, Industriestrasse 25, CH-9470 Buchs, Switzerland, e-mail: [email protected]. of Combinatorial Chemistry. Inst, of Molecular Biology and Genetics, NASU, P.O. 88, Kiev, 03187 Ukraine Sigma-Aldrich GmbH
Industriestrasse 25,CH-9470 Buchs, Switzerland
03302-661311
2D-Mini-Gels, 10µg E. Coli-extract, 7cm IPG-strips pH 3-10, 4-20% Tris-Gly Gel
LUCY-506 laser scanner, lex 473/filter 520
SYPRO Ruby laser scanner, lex 473/filter 580
Figure 2. Comparison between LUCY-506 and SYPRO Ruby in 2D-Electrophoresis.
Excitation and Emission spectra
0.00
0.20
0.40
0.60
0.80
1.00
1.20
220 270 320 370 420 470 520 570 620 670 720
[nm]
lex, max
LUCY-506 506 520LUCY-565 565 588LUCY-569 569 585
lem, max
Linear Dynamic Range Determination
Silver Stain
2000
1500
1000
500
0
Int-
bkg
1 32 61 92 122
153
183
214
245
Ovalbumin
BSA
Carboanhydrase
ng/band
Coomassie Stain
Ovalbumin
BSACarboanhydrase
1600
1400
1200
1000
800
600
400
200
0
Int-
bkg
ng/band
3
734
1464
2195
2925
3656
4386
5117
5847
Int-
bkg
ng/band
Int-
bkg
ng/band
SYPRO Red/LUCY-506/SYPRO Ruby(Carboanhydrase)
LUCY-569/LUCY-565/SYPRO Orange(Ovalbumin)
35000
30000
25000
20000
15000
5000
5000
0
3
369
734
1099
1464
1830
2195
2560
2925
3291
3656
SYPRO Red
LUCY-506
SYPRO Ruby
300000
250000
200000
150000
100000
50000
0
3
734
1464
2195
2925
3656
4386
5117
5847
LUCY-569
LUCY-565
SYPRO Orange
Figure 3. Dynamic range of LUCY stains in comparison to other staining methods.
Mass Spectrometry
Peptide mass fi ngerprint after in-gel-digest: 100ng E.Coli beta-galactosidase, separated by SDS-PAGE, stained with LUCY 506 (Spectrum A) and Sypro Ruby (Spectrum B), after band excision and trypsin-digest. Sequence coverage after LUCY-506 stain compared favorably to Sypro Ruby as determined by database analysis.
LUCY-506 SYPRO Ruby
A B100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
1000 1500 2000 2500 3000 3500 40001000 1500 2000 2500 3000 3500 4000 1000 1500 2000 2500 3000 3500 4000
m/zm/z
Figure 4. MALDI spectra of extracted peptides, crystallized with HCCA and measured on Shimadzu Kratos CFR MALDI-instrument in refl ectron mode.
LUCY-506 Bleaching (60min on UV-screen)
--
-
250
100
75 50 25 10 5 3 1
0 min 10 min 20 min 30 min 40 min 50 min 60 min
LUCY-506
min
LUCY-506laser scanner
lex 473 nm/lem 520 nm
Carboanhydrase 250 ngDifferenceBackground
-70% after 60 min
800
600
400
200
00 10 20 30 40 50 60
6645
29
--
-
6645
29
250
100
75 50 25 10 5 3 1 250
100
75 50 25 10 5 3 1 250
100
75 50 25 10 5 3 1 250
100
75 50 25 10 5 3 1 250
100
75 50 25 10 5 3 1 250
100
75 50 25 10 5 3 1
--
-
6645
29
--
-
6645
29
--
-
6645
29
--
-
6645
29
--
-
6645
29
SYPRO Ruby Bleaching (60min on UV-screen)
250
100
75 50 25 10 5 3 1
0 min 10 min 20 min 30 min 40 min 50 min 60 min
66 -45 -
29 -
Carboanhydrase 250ngDifferenceBackground
-83% after 60min
Sypro Rubylaser scanner
lex 473 nm/lem 580 nm
SyproRuby
min
600
500
400
300
200
100
00 10 20 30 40 50 60
250
100
75 50 25 10 5 3 1
250
100
75 50 25 10 5 3 1
250
100
75 50 25 10 5 3 1
250
100
75 50 25 10 5 3 1
250
100
75 50 25 10 5 3 1
250
100
75 50 25 10 5 3 1
66 -45 -
29 -
66 -45 -
29 -
66 -45 -
29 -
66 -45 -
29 -
66 -45 -
29 -
66 -45 -
29 -
Figure 5. Fluorescence intensity after 60min on a UV-screen. The intensity of the 250ng-Carboanhydrase-band is quantifi ed every 10min ( blue: Carboanhydrase; grey: background; red: difference)
Ordering InformationLUCY dyes / LUCY Starter KitLucy-506500 µl (5 mg/mL in DMSO)(Cat. No. 14149)
• lex 506 nm/lem 520 nm
• sensitivity comparable to SYPRO Orange
• all 3 dyes are compatible with mass-spectrometry
• all 3 dyes also stain native PAGEs after SDS-incubation
• better sensitivity than Coomassie
• better quantifi ability than silver
• no heavy-metals (SYPRO Ruby, silver)
• fast and easy staining protocol (no extra fi xation step); 1h total
• staining of gels with plastic-backing is possible (with reduced sensitivity)
• inexpensive
Lucy-565500 µl (5 mg/mL in DMSO)(Cat. No. 43772)
• lex 565 nm/lem 588 nm
• neutral staining conditions (allows western blot after gel staining)
Lucy-569500 µl (5 mg/mL in DMSO)(Cat. No. 41629)
• lex 569 nm/lem 585 nm
• broad linear dynamic range
Lucy Starter Kit3 x 50 µl (Cat. No. 04297)
• contains all 3 LUCY dyes
Dark Reader is a registered trademark of Clare Chemical Research, Inc. SYPRO is a registered trademark of Molecular Probes, Inc. Deep Purple is a trademark of GE Life Science.
Results
Detection Limits
SYPRO Orange 10-20% gel, laser scannerlex473 nm/Filter 520 nm
Silver Stain 10-20% gel, Dark Reader 535 nm/Filter 535 nm
SYPRO Red 10-20% gel, laser scannerlex532 nm/Filter 580 nm
SYPRO Tangerine10-20% gel, laser scannerlex473 nm/Filter 580 nm
SYPRO Ruby 10-20% gel, laser scannerlex473 nm/Filter 580 nm
Deep Purple 10-20% gel, laser scannerlex532 nm/Filter 580 nm
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d66
45
29
66
45
29
66
45
29
66
45
29
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d
Mar
ker
250
100
75 50 25 10 5 3 1 ng
/ban
d
LUCY-50610-20% gel, laser scannerlex473 nm/Filter 520 nm
LUCY-565 in water4-20% gel, laser scannerlex532 nm/Filter 580 nm
66
45
29
66
45
29
66
45
2966
45
29
Figure 1. Detection limits of LUCY and SYPRO stains as compared to other common protein stains. A protein standard mixture containing BSA (66 kDa), Ovalbumin(45 kDa) and Carboanhydrase (29 kDa) was used for all gel stains.