Far Eastern Blotting

Far eastern blotting is a simple technique in which the phospholipids and glycosphingolipids from a high- performance thin layer chromatography (HPTLC) plate is transferred to a pol yvinyl-diflouride (PVDF) membrane. It is also called TLC blotting. Most of the lipids developed on HPTLC plate is blotted quantitatively. Detection is done by either chemical or immunological staining. Purification of lipids is also possible in this method. Determination of lipid structure can also be carried out by coupling with mass spectrometry. Identification of enzymes and ligands of microorganisms also can be carried out TLC blotting / MS technique.

In signal transduction process lipids and their metabolites have an important part. These lipids are difficult to handle as secondary messengers in aqueous phase as they form micelle/ liposomes. They behave as large molecules with multivalency. The size of these large molecules vary with method of preparation and experimental conditions. Separation of the proteins which react specifically with particular lipids is exceedingly difficult. The challenges are countered by immobilizing the lipid to a solid phase. This immobilized lipid and the membrane mimics a biological membrane and provides convenient experimental conditions. It separates proteins which do not react with the immobilized lipids. Lipid purifying is difficult limiting lipid research techniques. Column chromatography coupled ith ion exchange and repeated silica bead columns have been used to purify lipid molecules. This process is time consuming. It requires a skillful techniques and use of toxic organic solvents.

TLC is the mostly used technique in detection, separation and monitoring of phospholipids and glycosphingolipids and some artificial chemicals and their metabolites.  TLC procedure with monoclonal antibodies is used in the structural analysis of glycosphingolipids. Silica gel used in the HPTLC plate has its own limitation, when to try to exchange the lipid from HPTLC plate there is a risk of silica gel contamination and sloughing off effect of silica gel during treatment is also a problem.  This limitations can be overcame if there is a transfer of lipids from HPTLC plate to a membrane having hydrophobic properties. Good separation of lipids takes place on HPTLC plate and if they are immobilized on a solid surface lipid studies can be made easier. Thus the attempt of lipid from HPTLC plate to the plastic membrane started. Nitrocellulose membrane were also used for the transfer but the transfer efficiency was poor and reproducibility factor was low. Various plastic membranes were tested and finally PVDF membrane suited the best. PVDF membrane is very stable against heating and various organic solvents. Also, the retaining efficiency of lipids on the PVDF is high.  Here is a procedure used for complex lipids.

The following are described:

  1. Method of TLC blotting,
  2. Microscale purification of complex lipids by TLC blotting,
  3. Direct mass spectrometric analysis of complex lipids on a PVDF membrane,
  4. Binding assay of microorganisms on a lipid immobilized membrane, and
  5. Lipid–protein interaction on lipid-immobilized membrane: ‘‘Simple method for detecting enzymes.’’

Method Of TLC Blotting

TLC Blotting of Glycosphingolipids

Glycosphingolipids are separated on a plate precoated with Silica Gel 60 (HPTLC plates). The plate is dried thoroughly with a hair dryer then dipped in the blotting solvent (isopropanol/ 0.2% aqueous CaCl2/ methanol, 40/20/7 by volume) for 20 s. A PVDF membrane and then a glass microfiber filter sheet are placed over the plate, and the whole assembly is pressed evenly for 30 s with a 180 0C iron. Most of glycosphingolipids have been transferred to the membrane.  Immersing the membrane in orcinol–H2SO4 reagent containing 50% (v/v) methanol and then heating it in an oven (100 0C)make the glycosphingolipid visible

The same procedure is used for blotting of the phospholipids to a PVDF membrane. The membrane is dipped in Dittmer–Lester reagent containing 50% (v/v) methanol, it makes the phospholipids visible. The color stain is so stable that it can remain for more than 1 year after staining, but the color on the HPTLC plate is quenched with time. Lipids transfer from the HPTLC plate to the PVDF membrane, depends linearly with the dose, for 0.1–3.0 g of each glycosphingolipid and for 2–10 g of each phospholipid.

Immunostaining on the PVDF Membrane

Immunostaining of antigen lipids can be done on the membrane. After blotting, the PVDF membrane was immersed in 1% bovine serum albumin in phosphate-buffered saline to block the nonspecific binding of protein. It is then incubated at 40C overnight in a nylon bag containing the first antibody. Peroxidase conjugated anti-mouse IgM antibody is used to detect the binding of the first antibody to the antigen lipid. Atleast 20 % of the antigen present in the HPTLC plate can be detected on the PVDF membrane. This indicates most of the glycosphingolipids transferred is concentrated on one side of the membrane and there is no chemical change occurs during the blotting process.

Microscale Purification Of Complex Lipids By TLC Blotting

Primuline reagent is used to visualize the glycosphingolipids or phospholipids separated on an HPTLC plate. This primuline reagent is very sensitive for the detection of lipid components. It does not change the native structure of the lipid. The bands are visible under UV light at 365 nm and is marked with a drawing pencil. These marks are also transferred to a PVDF membrane together with the lipids by TLC blotting. The blotted glycosphingolipids or phospholipids then are extracted from the marked areas.

There is a possibility of contamination of materials extracted from a PVDF membrane. To remove the contaminants, the extracted lipids are dissolved in 500 ml of chloroform: methanol (9:1, v/v) and then applied to a small silica beads column. The contaminants are eluted with chloroform:methanol (9:1, by vol) and lipids with 2 ml of chloroform:methanol:H2O (20:80:8, by vol). The yields of glycosphingolipids in our experiments ranged from 68.2 to 92.6%. The mean yield was 82.3%, and the yields of phosphatidylcholine and sphingomyelin were 90.4 and 83.4%, respectively.

Direct Ms Analysis Of Complex Lipids Transferred To A Pvdf Membrane (TLC BLOTTING/MS)

Mass spectrometric analysis can be used to characterize the structure of glycosphingolipids transferred to a PVDF membrane by TLC blotting. The marked glycosphingolipid area on the PVDF membrane is punched out and fitted to the probe tip of MS equipment. A few microliters of triethanolamine as a matrix is placed on the PVDF membrane, and negative MS spectra are obtained with a MS. This method is useful for the microscale analysis of glycosphingolipids in limited biological samples.

In the spectra, signals of the protonated molecular ions and fragmented ions are clearly visible. These signals, derived from fragmentation, make it possible to identify the sequences of sugar components and molecular species of ceramide.

TLC blotting/SIMS was used to analyze the glycosphingolipids of rat mammary tumor cell lines with different metastatic potentials for the lungs (MTLn2 with low metastatic potential and MTLn3 with high metastatic potential). All the glycosphingolipid bands (17 bands for MTLn2 and 13 bands for MTLn3) made visible by primuline reagent on an HPTLC plate, corressponding to the lipids obtained from 1 * 107 cells, were identified by this method.

Another application is the identification of incubation products of GalNAc-GD1a with sialidase in the presence of different activators, saposin B and GM2- activator protein. All the incubation products, which were separable only by TLC, could be identified, because the signals derived from fragmentation were clear enough to be assigned individual peaks. TLC blotting/ SIMS analysis of the incubation products clarified the mechanism of the activator in this enzyme reaction.

TLC/SIMS is useful for analyzing the structures of glycosphingolipids, but there are some problems. An especially designed mass spectrometer probe tip is needed. This device cannot be used with the samples developed by two-dimensional TLC. There also is the difficulty of cutting the HPTLC plate into small pieces. TLC blotting/SIMS overcomes these difficulties.

Binding Assay Of Microorganisms On A Lipid-Immobilized Membrane

Overlay binding assays on an HPTLC plate have been developed to determine glycosphingolipid ligands for bacteria. Although the procedure has provided much information there are some problems, such as the sloughing off of the silica gel from the plate during treatment. TLC blotting can be used for bacterial binding to glycosphingolipids. Moreover, the structural analysis of the ligand can be done using the TLC blotting/SIMS. We have named this procedure ‘‘in situ MS analysis of ligand.’’

The binding assay was done on the PVDF membrane using 35S-labeled Escherichia coli that carried K99 fimbriae (E. coli K99). Various glycosphingolipids separated on an HPTLC plate were transferred to a PVDF membrane. The membrane was blocked with PBS containing 4% casein and 0.1% methionine and then overlaid with 35S-labeled E. coli and incubated at 37 0C for 2 h. After the membrane was washed with PBS, binding was detected with a bioimaging analyzer. Radioactivity located on the band corresponded to such N-glycolyneuraminic acid-containing glycosphingolipids as sialylparagloboside, GM2, and GM3. The binding specificity of E.coli K99 to the glycosphingolipids was consistent with the findings of other investigators who used the overlay procedure on an HPTLC plate. These results indicate that the carbohydrate moieties of glycosphingolipids are exposed to the outer environment on the blotted membrane. Identification and molecular species analysis of ligand ganglioside was subjected to direct mass spectrometric analysis (in situ MS analysis). The ganglioside ligand from piglet intestine was identified as GM3 ganglioside with N-glycolylneuraminic acid and with ceramide species d18:1/C16h:0 from the clear fragmentation profile of the MS spectrum.

Lipid–Protein Interaction on the Lipid Immobilized Membrane

Two methods for detecting enzymes involved in glycosphingolipid metabolism on a PVDF membrane were developed. One is the detection of substrate glycosphingolipids on a PVDF membrane after TLC blotting. The glycosphingolipids on the membrane were incubated with an enzyme preparation, after which the product was detected by immunostaining with a monoclonal antibody directed to the product. The second one is the detection of enzymes located on a polyacrylamide gel. A sialidase preparation was electrophoresed on a polyacrylamide gel under nonreducing conditions and then transferred to a PVDF membrane impregnated with ganglioside as the enzyme substrate. The membrane then was incubated, and the product detected by immunostaining. The second method is principally based on the collagenase detection method, in which enzyme preparations are electrophoresed in a substrate- impregnated polyacrylamide gel, and the localization of the enzymes is detected after incubation.

These two methods basically depend on the detection of enzymes by immunoassay on the solid phase. Glycosidase treatment on HPTLC plates followed by detecting the products with monoclonal antibodies. A similar approach had been used to detect glycosyltrans ferase activity on HPTLC plates. To detect antibodies against glycosphingolipids in human sera, we developed a method to immobilize antigen glycosphingolipid on polystyrene beads. It also was used to purify specific antibodies to glycosphingolipid from antiserum. In this purification procedure, polystyrene beads and octylsepharose were used to support the glycosphingolipid. The immobilization of glycosphingolipids is based on the hydrophobic interaction between ceramide and the solid support, and this immobilization was used to detect enzyme activities. We developed an assay system for the detection of glycosidase and glycosyltransferase in a 96-well microtiter plate on which the substrate glycosphingolipid was immobilized. After incubation with enzyme, the amounts of product were determined by an enzyme immunoassay using a monoclonal antibody against the product. The method is simple, rapid, and specific for the detection of the product, and naturally occurring substrate can be used in the glycosidase assay. These advantages take care of the various difficulties encountered in using the enzyme assay for glycosyltransferase and glycosidase activities, but immobilization of the target lipid is solely dependent on the strength of the interaction between the solid phase and PVDF hydrophobic moiety of the lipid. Antigen glycosphingolipids frequently are released from a solid phase during incubation and repeated washing treatment. ELISA of autoantibody against gangliosides in human sera has been a major problemin obtaining reproducible results. To stabilize the immobilized lipids on the plastic plate, we treated it with polyisobutylmethacrylate, because most of the enzymes involved in glycosphingolipid metabolism require detergents for detection of their activities. We found that a PVDF membrane attached to a microtiter plate was best for obtaining stable immobilization of lipid on the solid phase. On the basis of these trials, we experimented with treating glycosphingolipid on a PVDF membrane with glycosidase and glycosyltransferase.

 Stability of the Glycosphingolipids on a PVDF Membrane 

Stability was assessed in the presence of detergent using Lc3Cer, nLc4Cer, and IV3NeuAcanLc4Cer. Release of these glycosphingolipids from the membrane to the aqueous phase (1 h incubation at 37oC) depended on the detergent concentration. At 0.01% Triton CF- 54, more than 95% of the glycosphingolipids were retained on the membrane. About 85% of the Lc3Cer and nLc4Cer were retained on the membrane at 0.05% Triton CF-54, whereas IV3NeuAcanLc4Cer was more easily released. Treatment of these glycosphingolipids with 5% detergent eluted more than 90% of the lipids to the aqueous phase.  Experiments on the time-dependent release from the membrane at 0.005% Triton CF-54 showed that all retained stable. After immersion of the membrane for 18 h at 37oC, more than 75% of the glycosphingolipids remained.

Sialidase Assay on a PVDF Membrane

Sialidase activity toward IV3NeuAcanLc4Cer transferred to a PVDF membrane was examined, using the  following conditions (1.0 munits of C. perfringens sialidase)/ml of 50 mM acetate buffer (pH 5.0) containing 0.005% Triton CF-54 and 0.5% BSA   in a nylon bag at 37oC for 1 h). After incubation, the  amount of nLc4Cer was determined by immunostaining.

Detection of Galactosyltransferase Activity toward Lc3Cer Transferred from an HPTLC Plate to a PVDF Membrane

Lc3Cer was developed on an HPTLC plate and the transferred to a PVDF membrane. The membrane was  placed in a nylon bag containing 2 ml of 25 mM cacodyl  ate buffer (pH 6.8) with 10 mM MnCl2 , 0.1 M KCl,  0.005% Triton CF-54, 1.56 mM UDP-galactose, and 1  munit of bovine milk galactosyltransferase. Incubation was done at 37oC for 30 min.  The amount of the incubation product, nLc4Cer, was assayed by immunostaining with the monoclonal antibody H11 followed by densitometric analysis.

Detection of Sialidase by Western Blotting to a Ganglioside-Impregnated PVDF Membrane

  1. perfringens sialidase (100 munit) dissolved in 20% glycerol containing 500 mM Tris–HCl buffer (pH 6.8) was electrophoresed on a 12.5% polyacrylamide slab gel, except that 0.005% Triton CF-54 was used instead of SDS. Afterward, the proteins were transferred electrophoretically to a V3NeuAcanLc4Cer-impregnated PVDF membrane. The membrane then was washed with water and incubated at 37oC for 1 h with 50 mM acetate buffer (pH 5.0) containing 0.005% Triton CF-54 and 0.1% BSA. The amount of nLc4Cer as the product of sialidase was determined by immunostaining with H11 MAb as described above. Silver staining showed that the sialidase preparation had some protein bands. One clear band was detected in the lane inwhich the enzyme preparation was blotted. The apparent molecular weight of the sialidase was estimated to be 99 kDa. nLc4Cer was detected when 1 munit of the enzyme was run through gel electrophoresis, whereas no band was detected in lane with 0.1 munit of the enzyme.

The same procedure was used to detect sphingomyelinase in a crude preparation of C. perfringens culture supernatant. In this case, [methyl-14C]sphingo-myelin was impregnated on a PVDF membrane. The electrophoretically separated sphingomyelinase was then transferred to the membrane by Western blotting. After incubation, the location of the enzyme was made visible by exposing the membrane to X-ray film.

Discussion

TLC is one of the easiest methods in lipid seperation. The advantages of TLC blotting are

  • It is simple and rapid (the entire procedure is completed within 1 min)
  • It gives high blotting efficiency
  • Detection of the blotted glycosphingolipids is more sensitive than it is on an HPTLC plate
  • The detection reagents for TLC can be used, and the colors on the membrane are stable. This procedure can also be used for transferring steroids and prostagladin3 with same blotting solvent mixture. The choice of blotting solvent determines the transfder of lipids from HPTLC plate to PVDF membrane. With right blotting solvent most of the lipid can be transferred to the PVDF membrane successfully. Radiolabeled nucleotide sugars are used for the assay of glycosyl transferase activity. First the reaction products is seperated with an organic solvent and the reaction products are identified on an HPTLC plate. The silica gel is scraped from the plate and its radioactivity is counted. In the case of glycosidases, artificial substrates are used to simplify the enzyme assay. The results obtained may sometimes slightly differ from those for the natural enzyme. This problem is counterec by introducing a new incubation system. This incubation system immobilizes glycosphingolipid substrates on a solid-phase microtiter plate and the amounts of product is determined using an enzyme immunoassay with a monoclonal antibody directed to the product. 125 I labeled toxin bound to GM1 is seperated by TLC blotting. Plastic membrane also was used for immunostaining glycosphingolipids.

TLC blotting and combined with immunostaining method is used for detecting glycosphingolipid-metabolizing enzymes. Enzyme activity can be determined with high sensitivity and specificity without using complicated separation processes.  Glycosphingolipids blotted on a PVDF membrane in the presence of detergent was stable. This allows to assay glycosidase and glycosyltransferase activities on a substrate-blotted membrane.  Western blotting from polyacrylamide gel to substrate-impregnated PVDF membrane is used for the detection of lipid metabolizing enzymes. This procedure sids in purification and detection of isozyme4 and inhibitors or activators. A lectin or toxin specific to reaction product can be used instead of a monoclonal antibody. Radiolabeled substrate can be used to detect phospholipase activities.