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TLC-Blot (Far-eastern Blot) and Its Application to MS Analysis of Glycosphingolipids
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TLC-Blot (Far-eastern Blot) and Its Application to MS Analysis of Glycosphingolipids

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Category
Glycolipids and related compounds
Protocol Name

TLC-Blot (Far-eastern Blot) and Its Application to MS Analysis of Glycosphingolipids

Authors
Taki, Takao *
-, AGT & T Co., Ltd.

Goto-Inoue, Naoko
Department of Marine Science and Resources, College of Bioresource Sciences, Nihon University
*To whom correspondence should be addressed.
KeyWords
Reagents

Solvent Systems for TLC developing

For neutral lipids:

Hexane/diethyl ether (70 : 30, v/v)

Petroleum ether/diethyl ether/acetic acid (70 : 30 : 1, v/v/v)

・For ceramides:

CHCl3/MeOH (95 : 5, v/v)

For phospholipids:

Methyl acetate/n-propanol/CHCl3/MeOH/0.25% KCl (25 : 25 : 25 : 10 : 9, v/v/v/v/v)

CHCl3/MeOH/0.2% CaCl2 (65 : 25 : 4, v/v/v)

・For gangliosides:

CHCl3/MeOH/0.2% CaCl2 (60 : 40 : 9, v/v/v) n-propanol/0.25% KCl (3:1, v/v)

Detection reagents for all lipid: primuline reagents

・Stock solution: One hundred mg of primuline is solved in 100 mL of water

・Spray reagent: One mL of the stock solution is diluted in 100 mL of a mixture of acetone/water (4:1, v/v)

Detection reagents for phospholipids, glycolipidsk, sialic acid-containing and amino group-containing lipids (see Notes)

Blotting solvent: isopropanol for TLC-Blot (Far-eastern Blot)/0.2% CaCl2/MeOH (40 : 20 : 7, v/v/v)

Matrix reagent (2,5-dihydroxybenzoic acid [DHB] for MALDI-TOF-MS: 50 mg/mL in MeOH/water (70 : 30, v/v)

Instruments

1. Thin-layer Chromatography (TLC)

TLC tank

Spray bottle

UV light (315 nm)

Safety glass

Syringe (10 μL)

Dryer

HPTLC plate (10 x 10 cm) ( Merck Millipore, Billerica, MA)

2. TLC-Blot (Far-eastern Blot)

Glass fiber filter membrane(AC-5972, 100 mm x 100mm: ATTO Corporation, Tokyo, Japan)

Teflon membrane (PIFE membrane, AC5973, 100mm x 100mm, ATTO)

PVDF membrane (Clear Blot membrane-P: ATTO)

TLC-Blotter (Nihon-Eido Co. Ltd. Tokyo, Japan)

3 TLC-Blot-MALDI-TOF MS

MALDI target plate

Electrified double-adhesive tape(P/N: 241-08728-92: Shimadzu Corporation, Kyoto Japan)

MALDI mass spectrometer

Methods
1.

Thin layer chromatography

1) 

 Before developing the plate, the TLC tank should be saturated with the developing solvent.

Comment 0
2) 

 One cm from the bottom of a HPTLC-plate, 5 mm-width marks are made with a soft pencil (6B) for applying the samples.

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3) 

 The lipid sample 5-10 mL is carefully and evenly applied on these pencil marks.

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4) 

 After applying the sample, the HPTLC plate is heated by a dryer for 1 min then developed in a TLC tank with the preferred developing solvent.

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5) 

 When the solvent front reaches about 7-8 cm from the bottom, the plate is taken and solvent on the plate is removed with a dryer.

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6) 

 The developed HPTLC plate is sprayed with primuline reagent.

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7) 

 After drying the plate, the lipid bands are visualized under UV light (315 nm). Use UV-cut glass for protection of the eyes.

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8) 

 The lipid bands are marked with a soft red-color pencil (so as not to damage the surface of the HPTLC plate).

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2.

TLC-Blot

1) 

 The plate is dipped in a blotting solvent for 10 sec.

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2) 

 Take the plate and cover the plate with a PVDF membrane immediately. Then, Teflon membrane and glass filter paper are mounted.

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3) 

 This assembly is transferred to a TLC-blotter that is heated at 180ºC in advance (Fig. 1). If this apparatus is not available, conventional heated iron is available for blotting.

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4) 

 Press the assembly for 30 sec to 1 min and take the PVDF membrane. Lipids separated on the HPTLC plate are transferred with red pencil marker. You will find the location of the lipids with the blotted pencil marks.

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3.

TLC Blot-MALDI-TOF MS

1) 

 Attach the PVDF membrane to MALDI target plate by electrified double-adhesive tape. Be sure to avoid air between MALDI target plate and PVDF membrane.

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2) 

 Apply matrix reagent to the band which was assigned by red pencil. Total of 500 μg DHB to 10 pmol of glycolipids is enough. After applying matrix, the PVDF membrane should be dry up.

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3) 

 Insert to mass spectrometer and get the signal on the band.

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4) 

 For imaging, we can choose raster mode analyses with automatic system. Fix the raster diameter, laser shot number, laser power, and area to be measured.

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5) 

 Imaging data (two-dimensional ion density map) are created using image reconstruction software (BioMap, Novartis, Basel, Switzerland). The ion image of each molecular weight can individually be constructed. In Fig. 2, we showed detection limit of TLC and TLC-Blot-MALDI-TOF MS. Moreover the distribution of the two molecular species of GM1 transferred on the PVDF membrane is scanned and exhibited as a fingerprinting profile.

Comment 0
Notes

Detection of phospholipids, glycolipids, sialic acid-containing lipids and amino group-containing lipids.

Molybdenum blue reagent (for phospholipid detection):

     A solution: Molybdenum three oxide 4.01 g is dissolved in 25N sulfuric acid by heating.

     B solution: Molybdenum powder 180 mg is added to 50 mL of A solution and the solution is heated for 15 min.

     After cooling, a clear upper solution was obtained by decantation.

     Spray reagent: The same volumes of A and B solution are mixed and the mixture is added to 2 volumes of water.

     The HPTLC plate is sprayed with the molybdenum blue (Dittmer reagent). After 5-10 min, a blue color remains on

     the bands of phospholipids.

 Orcinol reagent (for glycolipid detection):

     Orcinol 200 mg is solved in 11.4 mL of sulfuric acid then diluted to 100 mL with water. The HPTLC plate is sprayed

     with the orcinol reagent, and then the plate is heated on a hot plate at 105ºC for 5-10 min. Glycolipids are stained a purple color.

Resorcinol reagent (for detection of sialic acid-containing lipids):

     Resorcinol 200 mg is solved in 10 mL of water. Eighty mL of conc. HCl and 0.25 mL of 0.1M copper sulfate are added to the

     resorcinol solution and finally made up to 100 mL with water. The HPTLC plate is sprayed with the resorcinol reagent and

     covered with a glass plate.  The plate covered with a glass plate is heated on a hot plate at 105ºC for 5-10 min. Sialic acid-

     containing lipids are detected as blue-purple color bands.

Ninhydrin reagent (for amino group-containing lipid detection):

    Ninhydrin 300 mg is solved in 100 mL of 3% acetic acid-containing n-butanol. The HPTLC plate is heated at 100ºC for 10 min,

    and then the ninhydrin reagent is sprayed onto the plate.  Amino group-containing lipids are stained a pink color.

Figure & Legends

Figure & Legends

 

 

Fig. 1.   Scheme of TLC-Blot (far-eastern blot)/MALDI-TOF MS system

The figure is cited from ref. 1 with modification.

This figure was originally published in  Anal Biochem,Taki T. et al. "TLC blotting: application to microscale analysis of lipids and as a new approach to lipid-protein interaction"  251, 135-143,1997.

 

 

 

Fig. 2.  Representative results of TLC and TLC-Blot-MALDI-TOF MS of gangliosides.

Indicated amounts of GM1 separated on a HPTLC plate were detected with primuline staining then transferred to a PVDF membrane. Transfer of gangliosides separated on a HPTLC plate to PVDF membrane. Each GM1 band was directly analyzed by TLC-Blot-MALDI-TOF MS. The limit of detection is higher in TLC-Blot-MALDI TOF MS compared with primuline staining. Two distinct molecular species were separately visualized by imaging.

The figure is cited from Ref. 7 with modification.

This figure was originally published in J Chromatogr B Analyt Technol Biomed Life Sci., Taki T. et al "High-sensitivity analysis of glycosphingolipids by matrix-assisted laser desorption/ionization quadrupole ion trap time-of-flight imaging mass spectrometry on transfer membranes." 2008 Jul 1;870(1):74-83. doi: 10.1016/j.jchromb.2008.06.001. Epub 2008 Jun 6.

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