New Fluorescence Dyes For Protein Gel Stains
This document was uploaded by user and they confirmed that they have the permission to share it. If you are author or own the copyright of this book, please report to us by using this DMCA report form. Report DMCA
Overview
Download & View New Fluorescence Dyes For Protein Gel Stains as PDF for free.
More details
- Words: 1,478
- Pages: 1
New Fluorescence Dyes For Protein Gel Stains Bernhard Schönenberger1, Pierre Nording1, Sergiy Yarmoluk2, Alex Rück1, Monika Bäumle1, Michael Weber1 2
1 Sigma-Aldrich GmbH, Industriestrasse 25, CH-9470 Buchs, Switzerland, e-mail: [email protected] Dept. of Combinatorial Chemistry. Inst, of Molecular Biology and Genetics, NASU, P.O. 88, Kiev, 03187 Ukraine
Summary
Sigma-Aldrich GmbH Industriestrasse 25, CH-9470 Buchs, Switzerland
Results
Three new fluorescent protein gel stains, LUCY-506, LUCY-565 and LUCY-569 have been developed. The profile 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.
Mass Spectrometry Peptide mass fingerprint 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.
Detection Limits
1 ng/band
3
5
10
25
50
75
100
250
Marker
SYPRO Ruby
LUCY-506 100
1 ng/band
3
5
10
25
50
75
100
250
Marker
1 ng/band
3
5
10
25
50
75
100
250
Marker
1 ng/band
3
5
10
25
50
75
100
• 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 fluorescent 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 quantification on gel.
250
Marker
Motives
Deep Purple 10-20% gel, laser scanner lex532 nm/Filter 580 nm
Silver Stain 10-20% gel, Dark Reader 535 nm/Filter 535 nm
LUCY-565 in water 4-20% gel, laser scanner lex532 nm/Filter 580 nm
LUCY-506 10-20% gel, laser scanner lex473 nm/Filter 520 nm
90
100
A
90
80
80
70
70
60
60
50
50
40
40
30
30
20
66 66 66 45 29
66
10
0
0 1000
1500
2000
2500
45
29
45
20
10
45
B
3000
3500
4000
1000 1000
1500 1500
2000 2000
2500 2500 m/z
m/z
3000 3000
3500 3500
4000 4000
Figure 4. MALDI spectra of extracted peptides, crystallized with HCCA and measured on Shimadzu Kratos CFR MALDI-instrument in reflectron mode.
29
29
Staining Protocols
66 45 -
66 45 -
66 45 -
66 45 -
29 -
29 -
29 -
29 -
29 -
29 -
29 -
5 3
1
1
5 3
60 min 250 100 75 50 25 10
250 100 75 50 25 10
5 3
1
5 3
1
250 100 75 50 25 10
5 3
1
250 100 75 50 25 10
250 100 75 50 25 10
5 3
1
5 3
1
250 100 75 50 25 10
250 100 75 50 25 10
50 min
40 min
66 45 -
LUCY-506 800 600
Carboanhydrase 250 ng Difference Background
66
29
45
29
29
20
30
40
50
60
min
SYPRO Ruby Bleaching (60min on UV-screen)
Protein Mix
10 min
250 100 75 50 25 10 5 3 1
Gel Electrophoresis 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.
20 min
250 100 75 50 25 10 5 3 1
0 min
LUCY-565
10
30 min
50 min
40 min
60 min 250 100 75 50 25 10 5 3 1
29
0
250 100 75 50 25 10 5 3 1
45
66
LUCY-506 laser scanner lex 473 nm/lem 520 nm
0
-70% after 60 min
45
250 100 75 50 25 10 5 3 1
45
200
250 100 75 50 25 10 5 3 1
66
66
250 100 75 50 25 10 5 3 1
Detection Workflow
Gel-Stain
30 min
66 45 -
400
Table 1. Suitability and performance of LUCY stains for various staining procedures.
LUCY-506 LUCY-565 LUCY-569
20 min
66 45 -
1 ng/band
3
5
10
25
50
75
100
Marker
250
SYPRO Tangerine 10-20% gel, laser scanner lex473 nm/Filter 580 nm
1 ng/band
3
5
10
25
50
75
100
Marker
250
SYPRO Red 10-20% gel, laser scanner lex532 nm/Filter 580 nm
1 ng/band
3
5
10
25
50
75
100
250
Marker
1 ng/band
3
5
+++ -
10
+++ +++ +++
SYPRO Ruby 10-20% gel, laser scanner lex473 nm/Filter 580 nm
SYPRO Orange 10-20% gel, laser scanner lex473 nm/Filter 520 nm
25
+ ++ ++
+++ + +++
LUCY-506 Bleaching (60min on UV-screen) 10 min
0 min
50
Neutral stain before WB postelectrophoretic stain in water; time required: 1h; continue with Western-Blot transfer
75
Native PAGE stain run gel SDS-free; rinse gel in SDS; then standard procedure; time required: 1h 30min
100
Stain after fixation TCA-fixation; SDS-rinse; stain in NaOAc; time required: 1h 45 min
250
Prestaining procedure dye in cathoderunning-buffer with subsequent destaining in HOAc; time required: 15-60 min ++ + ++
Marker
LUCY-506 LUCY-565 LUCY-569
Standard procedure post-electrophoretic stain in HOAc; time required: 1h
66 45 -
66 45 -
66 45 -
66 45 -
66 45 -
66 45 -
66 45 -
29 -
29 -
29 -
29 -
29 -
29 -
29 -
Linear Dynamic Range Determination
2D-Mini-Gels, 10µg E. Coli-extract, 7cm IPG-strips pH 3-10, 4-20% Tris-Gly Gel
SyproRuby
SYPRO Ruby laser scanner, lex 473/filter 580
600
Coomassie Stain
Silver Stain 1600
2000
BSA
1000 Carboanhydrase
500
Laser Scanner
473 nm laser 532 nm laser
+++ -
Table 2. Imaging devices, methods and performance. 03302-661311
+++
++
0.00 220
ng/band
270
320
370
420
470 [nm]
520
570
620
670
5847
5117
4386
3656
720 Figure 3. Dynamic range of LUCY stains in comparison to other staining methods.
ng/band
5847
SYPRO Orange
0
5117
0.20
2925
2195
3
245
214
183
153
122
50000 4386
-
Lucy-569 500 µl (5 mg/mL in DMSO) (Cat. No. 41629)
100000
5000 0
0.40
30
40
50
60
Sypro Ruby laser scanner lex 473 nm/lem 580 nm
Ordering Information
150000
3656
+
SYPRO Ruby
20
LUCY dyes / LUCY Starter Kit Lucy-506 • lex 506 nm/lem 520 nm 500 µl (5 mg/mL in DMSO) • sensitivity comparable to (Cat. No. 14149) SYPRO Orange
LUCY-565
2925
-
15000 5000
200000
2195
+
LUCY-506
1464
+
20000
3
+++
520 588 585
734
0.60
590 nm band pass 590 nm band pass amber filter 520 nm cut off 580 nm cut off
506 565 569
Int-bkg
+++
++
LUCY-506 LUCY-565 LUCY-569
3656
0.80
LUCY-569
250000
10
min
Lucy-565 500 µl (5 mg/mL in DMSO) (Cat. No. 43772)
300000
3291
not tested
LUCY-569/LUCY-565/SYPRO Orange (Ovalbumin)
SYPRO Red
30000 25000
2925
not tested
0
Figure 5. Fluorescence intensity after 60min on a UV-screen. The intensity of the 250ng-Carboanhydrase-band is quantified every 10min ( blue: Carboanhydrase; grey: background; red: difference)
Ovalbumin
ng/band
2560
++
35000
2195
535 nm band pass 590 nm band pass 590 nm band pass amber filter 520 nm cut off 580 nm cut off
lex, max lem, max
1830
1.00
1464
not tested
734
not tested
1099
+
590 nm band pass 590 nm band pass amber filter 520 nm cut off 580 nm cut off
800 600
SYPRO Red/LUCY-506/SYPRO Ruby (Carboanhydrase)
369
Dark Reader® (lmax. ~450 nm)
1.20
orange filter
-
92
61
32
1 max.
Excitation and Emission spectra
Int-bkg
Imaging Illumination Device Polaroid Dark Reader® Camera (l max. ~450 nm) CCD UV screen Camera (l ~310 nm)
100 0
1000
ng/band
Optimum Detection Settings LUCY-569 Filter Sensitivity
200
0
0
Figure 2. Comparison between LUCY-506 and SYPRO Ruby in 2D-Electrophoresis.
LUCY-565 Filter Sensitivity
300
- 83% after 60min
400 200
MS
LUCY-506 Filter Sensitivity
Carboanhydrase
1464
Antibody Decoration
Int-bkg
Int-bkg
Membrane
Carboanhydrase 250ng Difference Background
BSA
1400 1200
Ovalbumin 1500
Band/Spot Excision Digest
500 400
734
LUCY-506 laser scanner, lex 473/filter 520
Western Blot
3
Imaging
Lucy Starter Kit 3 x 50 µl (Cat. No. 04297)
• lex 565 nm/lem 588 nm • neutral staining conditions (allows western blot after gel staining) • lex 569 nm/lem 585 nm • broad linear dynamic range • contains all 3 LUCY dyes
• all 3 dyes are compatible with mass-spectrometry • all 3 dyes also stain native PAGEs after SDS-incubation • better sensitivity than Coomassie • better quantifiability than silver • no heavy-metals (SYPRO Ruby, silver) • fast and easy staining protocol (no extra fixation step); 1h total • staining of gels with plasticbacking is possible (with reduced sensitivity) • inexpensive
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.
1 Sigma-Aldrich GmbH, Industriestrasse 25, CH-9470 Buchs, Switzerland, e-mail: [email protected] Dept. of Combinatorial Chemistry. Inst, of Molecular Biology and Genetics, NASU, P.O. 88, Kiev, 03187 Ukraine
Summary
Sigma-Aldrich GmbH Industriestrasse 25, CH-9470 Buchs, Switzerland
Results
Three new fluorescent protein gel stains, LUCY-506, LUCY-565 and LUCY-569 have been developed. The profile 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.
Mass Spectrometry Peptide mass fingerprint 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.
Detection Limits
1 ng/band
3
5
10
25
50
75
100
250
Marker
SYPRO Ruby
LUCY-506 100
1 ng/band
3
5
10
25
50
75
100
250
Marker
1 ng/band
3
5
10
25
50
75
100
250
Marker
1 ng/band
3
5
10
25
50
75
100
• 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 fluorescent 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 quantification on gel.
250
Marker
Motives
Deep Purple 10-20% gel, laser scanner lex532 nm/Filter 580 nm
Silver Stain 10-20% gel, Dark Reader 535 nm/Filter 535 nm
LUCY-565 in water 4-20% gel, laser scanner lex532 nm/Filter 580 nm
LUCY-506 10-20% gel, laser scanner lex473 nm/Filter 520 nm
90
100
A
90
80
80
70
70
60
60
50
50
40
40
30
30
20
66 66 66 45 29
66
10
0
0 1000
1500
2000
2500
45
29
45
20
10
45
B
3000
3500
4000
1000 1000
1500 1500
2000 2000
2500 2500 m/z
m/z
3000 3000
3500 3500
4000 4000
Figure 4. MALDI spectra of extracted peptides, crystallized with HCCA and measured on Shimadzu Kratos CFR MALDI-instrument in reflectron mode.
29
29
Staining Protocols
66 45 -
66 45 -
66 45 -
66 45 -
29 -
29 -
29 -
29 -
29 -
29 -
29 -
5 3
1
1
5 3
60 min 250 100 75 50 25 10
250 100 75 50 25 10
5 3
1
5 3
1
250 100 75 50 25 10
5 3
1
250 100 75 50 25 10
250 100 75 50 25 10
5 3
1
5 3
1
250 100 75 50 25 10
250 100 75 50 25 10
50 min
40 min
66 45 -
LUCY-506 800 600
Carboanhydrase 250 ng Difference Background
66
29
45
29
29
20
30
40
50
60
min
SYPRO Ruby Bleaching (60min on UV-screen)
Protein Mix
10 min
250 100 75 50 25 10 5 3 1
Gel Electrophoresis 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.
20 min
250 100 75 50 25 10 5 3 1
0 min
LUCY-565
10
30 min
50 min
40 min
60 min 250 100 75 50 25 10 5 3 1
29
0
250 100 75 50 25 10 5 3 1
45
66
LUCY-506 laser scanner lex 473 nm/lem 520 nm
0
-70% after 60 min
45
250 100 75 50 25 10 5 3 1
45
200
250 100 75 50 25 10 5 3 1
66
66
250 100 75 50 25 10 5 3 1
Detection Workflow
Gel-Stain
30 min
66 45 -
400
Table 1. Suitability and performance of LUCY stains for various staining procedures.
LUCY-506 LUCY-565 LUCY-569
20 min
66 45 -
1 ng/band
3
5
10
25
50
75
100
Marker
250
SYPRO Tangerine 10-20% gel, laser scanner lex473 nm/Filter 580 nm
1 ng/band
3
5
10
25
50
75
100
Marker
250
SYPRO Red 10-20% gel, laser scanner lex532 nm/Filter 580 nm
1 ng/band
3
5
10
25
50
75
100
250
Marker
1 ng/band
3
5
+++ -
10
+++ +++ +++
SYPRO Ruby 10-20% gel, laser scanner lex473 nm/Filter 580 nm
SYPRO Orange 10-20% gel, laser scanner lex473 nm/Filter 520 nm
25
+ ++ ++
+++ + +++
LUCY-506 Bleaching (60min on UV-screen) 10 min
0 min
50
Neutral stain before WB postelectrophoretic stain in water; time required: 1h; continue with Western-Blot transfer
75
Native PAGE stain run gel SDS-free; rinse gel in SDS; then standard procedure; time required: 1h 30min
100
Stain after fixation TCA-fixation; SDS-rinse; stain in NaOAc; time required: 1h 45 min
250
Prestaining procedure dye in cathoderunning-buffer with subsequent destaining in HOAc; time required: 15-60 min ++ + ++
Marker
LUCY-506 LUCY-565 LUCY-569
Standard procedure post-electrophoretic stain in HOAc; time required: 1h
66 45 -
66 45 -
66 45 -
66 45 -
66 45 -
66 45 -
66 45 -
29 -
29 -
29 -
29 -
29 -
29 -
29 -
Linear Dynamic Range Determination
2D-Mini-Gels, 10µg E. Coli-extract, 7cm IPG-strips pH 3-10, 4-20% Tris-Gly Gel
SyproRuby
SYPRO Ruby laser scanner, lex 473/filter 580
600
Coomassie Stain
Silver Stain 1600
2000
BSA
1000 Carboanhydrase
500
Laser Scanner
473 nm laser 532 nm laser
+++ -
Table 2. Imaging devices, methods and performance. 03302-661311
+++
++
0.00 220
ng/band
270
320
370
420
470 [nm]
520
570
620
670
5847
5117
4386
3656
720 Figure 3. Dynamic range of LUCY stains in comparison to other staining methods.
ng/band
5847
SYPRO Orange
0
5117
0.20
2925
2195
3
245
214
183
153
122
50000 4386
-
Lucy-569 500 µl (5 mg/mL in DMSO) (Cat. No. 41629)
100000
5000 0
0.40
30
40
50
60
Sypro Ruby laser scanner lex 473 nm/lem 580 nm
Ordering Information
150000
3656
+
SYPRO Ruby
20
LUCY dyes / LUCY Starter Kit Lucy-506 • lex 506 nm/lem 520 nm 500 µl (5 mg/mL in DMSO) • sensitivity comparable to (Cat. No. 14149) SYPRO Orange
LUCY-565
2925
-
15000 5000
200000
2195
+
LUCY-506
1464
+
20000
3
+++
520 588 585
734
0.60
590 nm band pass 590 nm band pass amber filter 520 nm cut off 580 nm cut off
506 565 569
Int-bkg
+++
++
LUCY-506 LUCY-565 LUCY-569
3656
0.80
LUCY-569
250000
10
min
Lucy-565 500 µl (5 mg/mL in DMSO) (Cat. No. 43772)
300000
3291
not tested
LUCY-569/LUCY-565/SYPRO Orange (Ovalbumin)
SYPRO Red
30000 25000
2925
not tested
0
Figure 5. Fluorescence intensity after 60min on a UV-screen. The intensity of the 250ng-Carboanhydrase-band is quantified every 10min ( blue: Carboanhydrase; grey: background; red: difference)
Ovalbumin
ng/band
2560
++
35000
2195
535 nm band pass 590 nm band pass 590 nm band pass amber filter 520 nm cut off 580 nm cut off
lex, max lem, max
1830
1.00
1464
not tested
734
not tested
1099
+
590 nm band pass 590 nm band pass amber filter 520 nm cut off 580 nm cut off
800 600
SYPRO Red/LUCY-506/SYPRO Ruby (Carboanhydrase)
369
Dark Reader® (lmax. ~450 nm)
1.20
orange filter
-
92
61
32
1 max.
Excitation and Emission spectra
Int-bkg
Imaging Illumination Device Polaroid Dark Reader® Camera (l max. ~450 nm) CCD UV screen Camera (l ~310 nm)
100 0
1000
ng/band
Optimum Detection Settings LUCY-569 Filter Sensitivity
200
0
0
Figure 2. Comparison between LUCY-506 and SYPRO Ruby in 2D-Electrophoresis.
LUCY-565 Filter Sensitivity
300
- 83% after 60min
400 200
MS
LUCY-506 Filter Sensitivity
Carboanhydrase
1464
Antibody Decoration
Int-bkg
Int-bkg
Membrane
Carboanhydrase 250ng Difference Background
BSA
1400 1200
Ovalbumin 1500
Band/Spot Excision Digest
500 400
734
LUCY-506 laser scanner, lex 473/filter 520
Western Blot
3
Imaging
Lucy Starter Kit 3 x 50 µl (Cat. No. 04297)
• lex 565 nm/lem 588 nm • neutral staining conditions (allows western blot after gel staining) • lex 569 nm/lem 585 nm • broad linear dynamic range • contains all 3 LUCY dyes
• all 3 dyes are compatible with mass-spectrometry • all 3 dyes also stain native PAGEs after SDS-incubation • better sensitivity than Coomassie • better quantifiability than silver • no heavy-metals (SYPRO Ruby, silver) • fast and easy staining protocol (no extra fixation step); 1h total • staining of gels with plasticbacking is possible (with reduced sensitivity) • inexpensive
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.
Related Documents
New Fluorescence Dyes For Protein Gel Stains
December 2019 5
Fluorescence
November 2019 9
Stains Comparison
May 2020 3
Modern Stains
November 2019 9
Gel
May 2020 15