ars.els-cdn.com€¦ · Web viewSupplemental table 1. Structural characterization of m/z 747. Detail of main fragments observed for fragmentation of m/z 747. The relative intensities

New visible and selective DNA staining method in gels with tetrazolium salts

Aaron J. Paredesa, Tatiana Naranjo-Palmaa, Hilda M. Alfaro-Valdésa, Andrés Barrigab, Jorge Babulc, Christian A.M. Wilsona,*

a Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santos Dumont 964, Independencia, Santiago, Chile.

b Unidad de Espectrometría de Masas-CEPEDEQ, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Sergio Livingstone 1007, Independencia, Santiago, Chile.

c Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Ñuñoa, Santiago, Chile.

Abbreviations: NBT, nitro blue tetrazolium; SGI, SYBR Green I; SGII, SYBR Green II; EB, Ethidium bromide; GR, gel red; SG, SYBR gold.

*Corresponding author. E-mail address: [email protected] (C.A.M. Wilson)

A

Supplemental figure 1. NBT-Dye staining method. The gel was soaked in a 1X of Dye (SG I, SG, GR or EB) and 0.20 mM of NBT in agitation for 25 min. Then it was exposed to sunlight for 90 min. After this process the purple bands appeared.

Visible without sunlight

Purple formazan precipitate

25 min

747.

1860

748.

1857

749.

1925

+MS

0.0

1.0

2.0

3.0

4.0

5.0

Inte

nsity

(î10

4 ) 6.0

A

747.

3185

748.

3191

749.

3262

750.

3281

751.

3370

752.

3524

0.0

0.5

1.0

1.5+MS

Inte

nsity

(î10

4 )

B74

7.5

748.

5

749.

5

+MS, 0.0-13.2min #(1-318)

0.0

0.2

0.4

0.6

0.8

740 745 750 755 760 765 m/z

Inte

nsity

(î10

5 )

C

Supplemental figure 2. Mass spectrometry analysis. A 12.5% non-denaturing polyacrylamide gel was loaded with 125 ng of DNA ladder and after electrophoresis the gels were stained with NBT-SGI and rinsed thrice with distilled water. About 45 DNA stained bands were cut with a scalpel and collected in a 2 mL vial with 1 mL of chloroform and stirred for 1 day at room temperature. The gels were then removed and the chloroform was extracted using a vacuum pump. The sample was suspended in 200 µL of tetrahydrofuran (THF) and then analyzed by mass spectrometry. A. MALDI-TOF mass spectrum acquired in positive mode in the CHCA matrix, a monoisotopic signal m/z 747.1860 was observed which could correspond to the monoformazan cation. B. MALDI-TOF mass spectrum acquired in positive mode in the 2.5-DHB matrix; monoisotopic signals m/z 747.1860 and m/z 749.3262 were observed which could correspond to the monoformazan cation and protonated form of diformazan respectively. C. ESI mass spectrum acquired in positive mode, a signal m/z 747.5 was observed and could correspond to the monocarbazan cation.

223.

9

419.

5

450.

446

4.5

477.

449

3.4

508.

3

596.

461

2.4

724.

2

751.

9

+MS2(747.5), 1.2-3.6min #(43-85)

0.0

1.0

2.0

3.0In

tens

ity(î

104 )

169.

7

213.

7

239.

825

6.1

342.

2

420.

0

447.

3

464.

3 477.

1

633.

4

+MS3(747.5->493.4), 5.2-5.5min #(118-122)

0.0

0.5

1.0

1.5

Inte

nsity

(î10

2 )

213.

7

419.

1

447.

1

0.00.51.01.52.02.5

Inte

nsity

(î10

3 )

+MS3(747.5->477.4), 2.6-2.8min #(69-72)

0 100 200 300 400 500 600 700 m/z

155.

5

172.

618

5.7

197.

821

1.6

257.

6

275.

129

1.1

308.

0

330.

2

406.

341

9.4

434.

9 562.

3

0.0

0.5

1.0

1.5

Inte

nsity

(î10

2 )

+MS3(747.5->464.5), 6.5-6.6min #(145-147)

Supplemental figure 3. Fragmentation analysis of m/z 747. ESI analysis for identification of the signal m/z 747, main fragments observed in MS2 spectrum were selected for MS3 fragmentation.

precursor (m/z)

fragments MSn (m/z) n

747.5 477.4 (100) 493.4 (67) 508.3 (24) 464.5

(24) 724.2 (19) 596.4 (12) 419.5 (11)

449.5 (7) 223.9

(5) 2

747.5→493.4 213.7 (100) 477.1 (45) 169.7 (20) 464.3

(18) 3

747.5→477.4 419.1 (100) 447.1 (14) 213.7 (4) 167.5

(3) 3

747.5→464.5 211.6 (100) 406.3 (32) 257.6 (24) 3

Supplemental table 1. Structural characterization of m/z 747. Detail of main fragments observed for fragmentation of m/z 747. The relative intensities of each fragment are indicated in parentheses.

A

N

N N

NH+

N O

O

m/z 268

N

N N

N

CH+

m/z 222N

N N

N

C+

m/z 221

-ƔNO2 -HNO2

N

N N

N+

OCH3 O CH3

N

N O

O

N

NNH

NO

O

m/z 747

N

NH

NH

N

OCH3

N N

NH2+

m/z 462

N

NH

NH

N

OCH3 O CH3

N N

NH2+

m/z 492

N

NH

NH

N

OH+

CH3

N N

NH

O

m/z 476

N

NH

NH

N N N

NH2+

O

m/z 446

N

NH

NH

N N N

NH2+

m/z 418

-CH4

-CH2=O

-COOH+

m/z 168

OCH3 OH+

CH3

m/z 213

N

NH

NH+

OCH3 O CH3

N N

NH

m/z 475

-2C7H5N3

-CH2=O

-CH2=O-NH3

N

N N

N

OCH3 O CH3

N N

NNH

OH+

m/z 596

NH

N N

N

OCH3 O CH3

N+

N O

O

N

m/z 508

OCH3 O CH3

N N

NNH

N+

O

OH

m/z 480

-C13H9N5O2

B

OCH3 O CH3

N N

NNH

N+

O

OH

m/z 480

O CH3

N N

NH+

NH

NO

O

O

m/z 464

N N

NH+

NH

NO

O

O O

m/z 448

CH+

N N

NNH

NO

O

O

m/z 420

O CH3

N N

NH+

NH

NO

O

O

m/z 465

O

N N

NNH

O

OH+

CH3

m/z 435

N N

NNH

O O

OH+

m/z 419

N N

NNH

O

OH+

m/z 405

OCH3 O CH3

NH+

N

NNH

C

NO

m/z 463

OCH3 OH+

CH3

m/z 211

O CH3

N N

NNH

C

N+

O

O

m/z 447

O CH3

N N

NNH

O

OH+

m/z 434

O CH3

N N

NNH

CH+

O

m/z 418

-ƔCH3

-ƔNO

-CH2=O-CH4

-CH4

-CO

-CH4

-ƔOH

-C13H10N5O

-ƔOH

-ƔNO2

-ƔNO

Supplemental figure 4. Proposed scheme of fragmentation for monoformazan. A, the scheme contains the main fragments observed in the different MS2 and MS3 spectra of m/z 747. B, the scheme described the fragmentation for m/z 480 and m/z 464. Fragmentation proposal contains virtually all experimentally observed fragmentations and explains the MS2 and MS3 spectra.

Supplemental figure 5. XbaI enzymatic cut assay. The pDJ100 plasmid was loaded in a 0.8% agarose gel and run at 100 V for 1 h in TAE 1X. Then gel was stained with: SG, NBT-SGI, Ctrl UV (+) and Ctrl UV (-). Finally, the gel band were extracted using a gel extraction kit. After the extraction, the plasmid was subjected to an enzymatic cut with XbaI enzyme. Then, the treated plasmid was run in a 5% agarose gel in TAE 1X at 100 V for 2 h. Finally the gel was stained with SG. The figure shows the cut percentage after extraction and XbaI treatment for the different staining methods. The wells are: 1) ladder DNA, 2) cut NBT-SGI treated plasmid, 3) non-cut NBT-SGI, 4) cut ctrl UV (-), 5) non-cut ctrl UV (-), 6) cut SGI, 7) non-cut SGI, 8) cut Ctrl UV (+), 9) non-cut Ctrl UV (+). All staining procedures were carried out with n=3.

1 2 3 4 5 6 7 8 9

A

B

Supplemental figure 6. A. Specific wavelength. DNA-SGI complex absorption spectra (solid line) was downloaded from Thermo-Fisher. NBT absorption spectra (dashed line) was obtained using a Hewlett Packard 8453 spectrophotometer at 0.2 mM. Blue LED (dot line) was obtained using a High-Resolution Spectrometer HR2000, Ocean Optics (Dunedin, FL, US). B. Precipitation of formazan by blue light illumination. The gel was soaked in a 1X of SGI and 0.20mM of NBT in agitation for 25 min. Then it was exposed to blue LED for 5 min. After this process, the purple bands appeared.

Visible without blue light

Blue LED

Purple formazan precipitate

5 min

NBT + Dye 25 min

Thermo Fisher Scientific. Fluorescence SpectraViewer [on line]. Retrieved from https://www.thermofisher.com/cl/es/home/life-science/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html?ICID=svtool&UID=7567dna