Total lipid extracts were analyzed by TLC on silica gel 60A plates (Merck, 20 × 10 cm, layer thickness, 0.2 mm). The plates were washed two times with chloroform/methanol (1:1, by vol.) and activated at 180°C before use. Polar lipids were eluted with an acid solvent (chloroform/methanol/acetic acid/water, 85:15:10:3.5, by vol.). Total lipid detection was carried out by molybdenum blue spray reagent (Sigma-Aldrich) specific for phospholipids (Kates, 1986 ). Alternatively, total lipid detection was done with reversible staining exposing the TLC plate to iodine vapor for 4–5 min for staining all classes of lipids before the lipid bands isolation. To analyze in detail the various components of the lipid extracts, each band present on the plates was scraped and lipids extracted from silica, as previously described (Kates, 1986 ); briefly, 0.5 ml of a mixture chloroform/methanol/water (1:2:0.8, by vol.) has been added to silica bands, and the samples were vigorously stirred and centrifuged. Lipid bands of preparative TLC were analyzed by positive and negative ion mode MALDI-TOF/MS.
Molybdenum blue spray reagent
Manufactured by Merck Group
Sourced in United States
Molybdenum blue spray reagent is a chemical compound used for the detection and quantification of phosphate ions in various samples. It is a sensitive and reliable reagent for colorimetric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Silica gel 60a plates, by Merck Group (2 mentions)
Automatic developing chamber adc 2, by CAMAG (1 mentions)
Linomat 5 sample application unit, by CAMAG (1 mentions)
Sulfuric acid, by Merck Group (1 mentions)
Silica gel 60, by Merck Group (1 mentions)
Ninhydrin, by Merck Group (1 mentions)
Hp 6890 series gc system, by Agilent Technologies (1 mentions)
Zaragozic acid, by Merck Group (1 mentions)
1 1 dioctadecyl 3 3 3 3 tetramethylindodicarbocyanine did, by Thermo Fisher Scientific (1 mentions)
Carbonyl cyanide m chlorophenyl hydrazone cccp, by Abcam (1 mentions)
1 2 dioleoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phospho l serine, by Avanti Polar Lipids (1 mentions)
Egg l α phosphatidylcholine, by Avanti Polar Lipids (1 mentions)
Adamant tlc plates, by Macherey-Nagel (1 mentions)
Silica gel 60 tlc plate, by Merck Group (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine n methyl, by Avanti Polar Lipids (1 mentions)
1 2 dioleoyl sn glycero 3 phosphoethanolamine n n dimethyl, by Avanti Polar Lipids (1 mentions)
8 protocols using molybdenum blue spray reagent
1
Comprehensive Lipid Analysis by TLC-MALDI
2
Lipid Extraction and Quantification Protocol
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Polar lipids were extracted from exponential-phase bacteria with chloroform-methanol (2:1 [by volume]) by the Bligh-Dyer procedure (33 (link)), vacuum dried, and dissolved in chloroform-methanol (2:1 [by volume]). For detection of Lys-PG, appropriate amounts of polar lipid extracts were spotted onto silica gel 60 F254 high-performance thin-layer chromatography (HPTLC) plates (Merck, Darmstadt, Germany) using a Linomat 5 sample application unit (Camag, Berlin, Germany) and developed with chloroform-methanol-water (65:25:4 [by volume]) in an automatic developing chamber ADC 2 (Camag, Berlin, Germany). Lys-PG content was quantified as described recently (6 (link)). Phospholipids of exponentially growing strains were selectively stained with molybdenum blue spray reagent (1.3% molybdenum oxide dissolved in 4.2 M sulfuric acid [Sigma]). Integrated lipid spot intensities of molybdenum blue-stained phospholipids were determined with ImageJ (http://rsbweb.nih.gov/ij/ )
3
Phospholipid Analysis and Membrane Fractionation in Bacteria
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Bacteria were grown in LB to an optical density at 650 nm (OD650) of ~0.5 (mid-log phase) or as indicated. Phospholipids were extracted by the method of Bligh and Dyer (44 (link)). Phospholipids were then spotted onto a silica gel 60 (Millipore) thin-layer chromatography (TLC) plate and separated by TLC using a chloroform-methanol-acetic acid (65:25:10, vol/vol/vol) solvent system (67 (link)). Phospholipids were detected by spraying TLC plates with molybdenum blue spray reagent (Sigma). The area of species was quantified using ImageJ (68 (link)) to determine the percentage of each phospholipid in the sample.
Inner and outer membranes were isolated by pelleting mid-log-phase bacteria at 13,000 × g for 10 min, resuspending in buffer containing 10 mM Na2HPO4 and 5 mM MgSO4, sonicating to induce cell lysis, and centrifuging at 13,000 × g for 20 min to remove cell debris. The supernatant was then centrifuged at 135,000 × g for 40 min to isolate the total membranes. Total membranes were resuspended in 1.0% (wt/vol) Sarkosyl using a blunt needle and incubated at room temperature for 20 min. Following centrifugation at 135,000 × g for 40 min, the inner membranes remained in the supernatant, while the outer membranes were pelleted. The pelleted outer membranes were resuspended in fresh 1.0% (wt/vol) Sarkosyl and used for downstream assays.
Inner and outer membranes were isolated by pelleting mid-log-phase bacteria at 13,000 × g for 10 min, resuspending in buffer containing 10 mM Na2HPO4 and 5 mM MgSO4, sonicating to induce cell lysis, and centrifuging at 13,000 × g for 20 min to remove cell debris. The supernatant was then centrifuged at 135,000 × g for 40 min to isolate the total membranes. Total membranes were resuspended in 1.0% (wt/vol) Sarkosyl using a blunt needle and incubated at room temperature for 20 min. Following centrifugation at 135,000 × g for 40 min, the inner membranes remained in the supernatant, while the outer membranes were pelleted. The pelleted outer membranes were resuspended in fresh 1.0% (wt/vol) Sarkosyl and used for downstream assays.
4
Bacterial Cell Characterization Techniques
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Cell morphology was determined by transmission electron microscopy (JEM-1400, jeol ). The utilization of carbon sources was determined using the 96-well Biolog AN MicroPlate that contained 95 different carbon substrates [34 (link)]. Bacteria strains were cultured in liquid mGAM medium for 2 days, then cells were harvested. Cellular fatty acids were extracted and methylated according to the standard midi protocol (Sherlock Microbial Identification System, version 6.0). The identification was performed by GC (HP 6890 Series GC System; Agilent) [35 ]. Polar lipids were separated by two-dimensional thin-layer chromatography (TLC plates coated with silica gel, 1010 cm; Merck). Chromatography was performed using chloroform–methanol–water (65 : 25 : 4, by vol.) for the first dimension, followed by chloroform–methanol–acetic acid–water (80 : 12 : 15 : 4, by vol.) for the second dimension [36 (link)]. Total lipids were detected with 10 % ethanolic molybdatophosphoric acid (Sigma). Aminolipids were detected with 0.4 % solution of ninhydrin (Sigma) in butanol. Phospholipids were detected with Zinzadze reagent (molybdenum blue spray reagent, 1.3 %; Sigma) and glycolipids were detected with 0.5 % α-naphthol sulphuric acid reagent.
5
Lipid Extraction and Characterization by TLC
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Total lipid extracts were analyzed by thin layer chromatography (TLC) on silica gel 60 A plates (Merck, 20 × 10 cm, layer thickness 0.2 mm). The plates were washed twice with chloroform/methanol (1:1, by volume) and activated at 180 °C before use. Polar lipids were eluted with Solvent A (chloroform/methanol/acetic acid/water 85:15:10:3.5, by volume).
Lipid detection was carried out by spraying the plate with 5% sulfuric acid in water, followed by charring at 180 °C for 5 min; moreover, the following stainings were performed in order to identify the lipid classes present in the TLC bands: (i) molybdenum blue spray reagent (Sigma-Aldrich) specific for phospholipids, and (ii) ninhydrin solution, prepared dissolving 0.25 g of reagent grade ninhydrin in 100 ml of acetone-lutidine (9:1, by volume), for phosphatides or lipids having a free amino group48 .
Lipid detection was carried out by spraying the plate with 5% sulfuric acid in water, followed by charring at 180 °C for 5 min; moreover, the following stainings were performed in order to identify the lipid classes present in the TLC bands: (i) molybdenum blue spray reagent (Sigma-Aldrich) specific for phospholipids, and (ii) ninhydrin solution, prepared dissolving 0.25 g of reagent grade ninhydrin in 100 ml of acetone-lutidine (9:1, by volume), for phosphatides or lipids having a free amino group48 .
6
Lipid Membrane Composition Analysis
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E. coli CL, E. coli L-α-PG, egg L-α-phosphatidylcholine (eggPC), E.coli L-α-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). 1-(4-Trimethylammoniumphenyl)-6-Phenyl-1,3,5-Hexatriene p-Toluenesulfonate (TMA-DPH) and N-(3-Triethylammoniumpropyl)-4-(6-(4-(Diethylamino) Phenyl) Hexatrienyl) Pyridinium Dibromide (FM4-64), and 1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindodicarbocyanine (DiD) were from Thermo Fisher Scientific (Waltham, MA, USA). Molybdenum Blue spray reagent was from Sigma-Aldrich (Saint Louis, MI, USA). Carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was purchased from Abcam (Branford, CT, USA), and valinomycin was purchased from VWR International (Radnor, Pennsylvania, USA). 3-(N-maleimidylpropionyl)biocytin (MBP) was obtained from Invitrogen (Waltham, MA, USA) and the HRP-conjugated antibody from eBioscience (San Diego, CA, USA). Zaragozic acid was purchased from Sigma-Aldrich. 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) and Zaragozic acid were from obtained from Cayman Chemical (Ann Arbor, MI, USA).
7
Mitochondrial Phospholipid Extraction and Quantification
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Phospholipid extraction, staining, and quantitation were performed essentially as described15 (link). In brief, phospholipids were extracted from 1 mg of mitochondria with chloroform:methanol, loaded onto ADAMANT TLC plates (Machery-Nagel), and resolved once in chloroform:ethanol: H2O:triethylamine (30:35:7:35). Phospholipids were visualized using a 1.3% molybdenum blue spray reagent (Sigma). Phosphate concentration of extracted lipids was performed as described55 (link).
8
Lipid Isolation and Characterization
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2-(methylamino)ethanol (MMEA), 2-(ethylamino)ethanol (DMEA), 1-oleoyl-2-hydroxy-sn-glycero-3-phosphocholine sodium salt (LPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (PG), 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC), 1′,3′-bis(1,2-dioleoyl-sn-glycero-3-phospho)-glycerol (CL), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-methyl (MMPE), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N,N-dimethyl (DMPE) were purchased from Avanti polar lipids. TLC silica gel 60 plates and molybdenum blue spray reagent were purchased from Sigma-Aldrich. All other chemicals used were of analytical grade and commercially available.
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