COST-EFFECTIVE AND EFFICIENT APPARATUS FOR ELECTROELUTION OF MICRO- AND MACROGRAM QUANTITIES OF PROTEINS FROM POLYACRYLAMIDE GELS

Protein recovery from gel electrophoresis plays a significant role in functional genomics and proteomics. To assist in this, a simple, cost-effective, and efficient apparatus for electroelution of proteins has been designed. The performance of the apparatus was demonstrated using the proteins bovine serum albumin (BSA), phosphorylase, ovalbumin, pepsin, and trypsinogen. In all the cases the yield of elution was found to be consistently greater than 85% and the proteins could be eluted without degradation in less than 15 min. The utility of this method can be extended to protein elution from denatured and native polyacrylamide gels, DNA purification from agarose gels, and oligomeric primers purification from polyacrylamide gels. In addition to this, the method offers an effortless purification and characterization of microbial extracellular proteins. The eluted proteins can be directly used in N-terminal amino acid sequencing, and in amino acid and proteomics analyses.     

Purification, amino terminal analysis, and peptide mapping of proteins after in situ postelectrophoretic fluorescent labeling

Proteins fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were stained in situ with either 5-(dimethylamino)-1-naphthalene sulfonyl chloride (dansyl chloride) or fluorescein isothiocyanate. This staining procedure can be carried out in less than 30 min without previous fixation of the proteins. It is not dependent on such factors as charge or molecular weight of the proteins and can detect 50 ng of protein in a 10-mm-wide gel slot. Fluorescent staining with dansyl chloride was used to localize proteins after electrophoresis for subsequent electroelution, amino terminal analysis, and peptide mapping. The electroelution can be carried out in less than 3 h with yields approaching 100%. The staining of only one strip of a preparative gel allowed the electroelution of proteins without covalent modification. For amino terminal analysis, identical results were obtained when the hydrolysis step was carried out after electroelution or directly in the gel pieces. The peptide mapping can be carried out with the proteins in solution (after electroelution) or directly in the gel pieces. The amino terminal and peptide mapping analysis of each protein in a mixture can be completed within 30 h from the beginning of the electrophoretic fractionation. The method appears to be applicable to a wide range of proteins showing very different biochemical properties.     

Transfer of SDS-proteins from gel electrophoretic zones into mass spectrometry, using electroelution of the band into buffer without sectioning of the gel

Five SDS-proteins, ranging in molecular weight from 14 to 66 kDa, were detected without covalent fluorescent labeling by the automated gel electrophoresis apparatus with intermittent fluorescence scanning (HPGE apparatus, LabIntelligence) during electrophoresis in barbiturate buffer in the presence of Cascade Blue. The SDS-proteins were electroeluted from the gel into 220 microl of buffer by a modification of the procedure of Gombocz and Cortez. The electroeluate was freed of SDS, ultrafiltered and subjected to MALDI-TOF mass spectrometry. The masses of the five native proteins were found to be maintained after electrophoresis and electroelution in the presence of the potential contaminants SDS, barbituric acid and Cascade Blue. The procedure of protein transfer from SDS-PAGE into mass spectrometry, without excision of bands, gel maceration and protein recovery by diffusion, therefore is shown to be suitable for the identification by mass of intact proteins derived from gel electrophoretic bands.     

Recovery of sodium dodecyl sulfate-proteins from gel electrophoretic bands in a single electroelution step for mass spectrometric analysis

Mass spectrometric analysis of proteins derived from bands in gel electrophoresis is incompatible with the covalent fluorescent labeling of the protein. Thus, if one wishes to take advantage of the capacity for computer-directed electroelution of electrophoresis apparatus with intermittent fluorescent scanning of the migration path, the protein must be labeled fluorescently in a noncovalent, reversible fashion. This was recently achieved by staining of SDS-proteins with Cascade blue and electrophoresis in barbital buffer. However, the method was not a practical one for the purpose of isolating proteins from gel electrophoretic bands and their transfer into the mass spectrometer for three reasons: (i) Ten consecutive electroelution steps were required to obviate pH changes in the electroelution chamber; (ii) electroeluates from six gel electrophoretic lanes needed to be pooled; (iii) excessive protein loads ranging from 7 to 33 microg/pool were required. The present study reports the solution to those three problems. Mass spectrometric (MALDI-TOF) characterization of five proteins was demonstrated (i) after a single electroelution step; (ii) using electroelution from a single gel of 0.3-cm(2) cross-sectional area; and (iii) using a protein load of 2 (in one case 4) microg. However, the migration rates of the Cascade blue-SDS-protein-barbital complexes derived from proteins with widely varying molecular weights proved to be the same. Thus, despite the three advances made, the method to date remains restricted to samples of single proteins.     

Isolation and purification of microbial community DNA from soil naturally enriched for chitin

We made an attempt to isolate and purify metagenomic DNA from chitin enriched soil. In this communication we report a modified direct lysis method for soil DNA extraction including initial pre-lysis washing of sample, followed by a rapid polyvinylpyrrolidone-agarose-based purification and electroelution of DNA using Gene-capsule™ assembly. Rapidity was achieved using low molarity conducting media (sodium-borate buffer) for electrophoresis by reducing run time for both the gel electrophoresis and electroelution. Extracted DNA was sufficiently pure and of high quality, evidenced by amplification of 16S rDNA and chitinase genes by PCR. Metagenomic nature of the DNA was confirmed by running V3 (16S rDNA) region amplicons using denaturing gradient gel electrophoresis. This method requires 30 min for purification, and less than 2 h for complete execution of protocol and becomes the first report on the isolation of metagenomic DNA from soil naturally enriched for chitin.     

Identification and characterization of the Sulfolobus solfataricus P2 proteome

Via combined separation approaches, a total of 1399 proteins were identified, representing 47% of the Sulfolobus solfataricus P2 theoretical proteome. This includes 1323 proteins from the soluble fraction, 44 from the insoluble fraction and 32 from the extra-cellular or secreted fraction. We used conventional 2-dimensional gel electrophoresis (2-DE) for the soluble fraction, and shotgun proteomics for all three cell fractions (soluble, insoluble, and secreted). Two gel-based fractionation methods were explored for shotgun proteomics, namely: (i) protein separation utilizing 1-dimensional gel electrophoresis (1-DE) followed by peptide fractionation by iso-electric focusing (IEF), and (ii) protein and peptide fractionation both employing IEF. Results indicate that a 1D-IEF fractionation workflow with three replicate mass spectrometric analyses gave the best overall result for soluble protein identification. A greater than 50% increment in protein identification was achieved with three injections using LC-ESI-MS/MS. Protein and peptide fractionation efficiency; together with the filtration criteria are also discussed.     

Electroelution of protein fractions from a polyacrylamide gel

A method for electroelution of protein fractions from polyacrylamide gel and device for performing such a process have been developed. The application of two tris-glycine buffers with the low and high ionic strength, pH 9.0-9.2 provides a concentration of protein simultaneously to extraction from the gel. The duration of elution is in the range of 1-3 hours and depends on the protein mobility. The effectiveness of the system is demonstrated for disc-electrophoretic separation and electrophoresis in slab gel in the presence of SDS. The maximal amount of pure protein fraction obtained is about 4.5-5.0 mg. The method may be useful especially for the fractionation of limited quantities of protein samples.     

Depletion of hemoglobin and carbonic anhydrase from erythrocyte cytosolic samples by preparative clear native electrophoresis

Proteomic analysis of red cells is compromised by the presence of high-abundance proteins (hemoglobin and carbonic anhydrase-1), which completely obscure low-abundance species. The depletion method presented here involves performing native gel electrophoresis in a polyacrylamide gel tube using a modified electroelution cell. The electrophoretic run is interrupted intermittently to allow the recovery of at least three different liquid fractions, which can be analyzed by both native PAGE and 2D isoelectric focusing SDS-PAGE, or by shotgun mass spectrometry analysis after trypsin in-solution protein digestion. This low-cost, reproducible technique can be used to process large amounts of sample, and it increases the likelihood of detecting low-abundance proteins, thereby resulting in greater proteome coverage. The separation procedure takes approximately 6-7 h.     

Manual N-terminal microsequencing of proteins electroeluted from polyacrylamide gel slices

A simple and rapid procedure for preparation of proteins for manual microsequencing using sodium dodecyl sulfate gel electrophoresis is described. The procedure involves pre-electrophoretic labeling of the protein amino groups with a coloured Edman reagent, disk electrophoresis for purification or fractionation of the proteins, and reversed electrophoretic transfer of the separated protein from gel slices into a small volume of buffer (100 to 150 microliter) using a discontinuous conductivity gradient to recover the proteins. The pre-electrophoretic labeling facilitates location of the separated proteins in the gel and the monitoring of their complete electroelution. The isolated proteins are separated from excess of salts by acetone precipitation and solvent partitioning in pyridine/water (1:1) and subjected to manual DABITC/PITC degradation.     

Diffusion-based extraction of DMSO from a cell suspension in a three stream, vertical microchannel

Cells are routinely cryopreserved for investigative and therapeutic applications. The most common cryoprotective agent (CPA), dimethyl sulfoxide (DMSO), is toxic, and must be removed before cells can be used. This study uses a microfluidic device in which three streams flow vertically in parallel through a rectangular channel 500 μm in depth. Two wash streams flow on either side of a DMSO-laden cell stream, allowing DMSO to diffuse into the wash and be removed, and the washed sample to be collected. The ability of the device to extract DMSO from a cell stream was investigated for sample flow rates from 0.5 to 4.0 mL/min (Pe = 1,263-10,100). Recovery of cells from the device was investigated using Jurkat cells (lymphoblasts) in suspensions ranging from 0.5% to 15% cells by volume. Cell recovery was >95% for all conditions investigated, while DMSO removal comparable to a previously developed two-stream device was achieved in either one-quarter the device length, or at four times the flow rate. The high cell recovery is a ~25% improvement over standard cell washing techniques, and high flow rates achieved are uncommon among microfluidic devices, allowing for processing of clinically relevant cell populations.     

Fast electrotransfer of human serum proteins in their native state from polyacrylamide thin gradient gels reinforced by textiles to polyvinylidene difluoride membranes. Rapid electrotransfer from thin gradient gels reinforced by textiles.

A fast electroblotting technique of native molecules electrophoretically separated in thin (0.25 to 0.5 mm) gradient gels, onto a high capacity membrane of polyvinylidene difluoride is described. Omitting methanol during transfer, the equilibration step is avoided and the same buffer is used in electrophoresis and transfer. As the gel reinforced by fabric never swells nor shrinks, and as all the bands are blotted, the transfer matrix exactly reflects the protein pattern of the original gel. Autoradiography is enhanced and electroelution is homogeneous in all parts of the gels. Significant improvement is noticed in binding proteins of molecular weight from about 20 kDa to more than 700 kDa, as suggested by complete electroelution of all native serum components.     

Single-step electroelution of proteins from SDS-polyacrylamide gels and immobilization on diisothiocyanate-glass beads in prepacked capillary columns for solid-phase microsequencing

A new method for immobilization of proteins purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) prior to sequencing is described. It utilizes a simple apparatus that permits the simultaneous electroelution of proteins from gel slices and attachment to diisothiocyanate-activated glass beads prepacked in capillary tubes [S-P. Liang and R. A. Laursen, Anal. Biochem. 188, 366-373 (1990)]. Transfer/attachment yields of greater than 80% within 90 min were observed for several 125I-labeled proteins with a range of molecular weights using 0.2 M sodium phosphate (pH 8.9) buffer containing 0.1% SDS. The method has the advantage of high capacity, relative simplicity, and insensitivity to the presence of SDS and Coomassie blue stain. The highest transfer yields were obtained when proteins were run on gels which had been aged for at least 12 h. For 100- to 1000-pmol samples, the sequenceable amount of protein, including transfer, was generally 30-60%, with an average repetitive yield of 95%. Factors which influence sample recovery and sequencing yield are discussed.     

Further development of an electroosmotic medium pump system for preparative disk gel electrophoresis

A simple and practical 6.8-cm-diameter (36.30-cm(2) cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate-PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.     

Potential of Aqueous Two-Phase Systems constructed on flexible devices: Human serum albumin as proof of concept

In the present research, the potential use of flexible disposable devices, specifically blood bags, for the fractionation of biological products using Aqueous Two-Phase Systems (ATPS) polymer–salt is studied and demonstrated. Purified human serum albumin (HSA) was used as model protein. Experiments were carried out on ATPS polyethylene glycol (PEG)–potassium phosphate constructed on rigid recipients (conical tubes) and flexible devices (blood bags). The device used for ATPS construction had no significant effect on HSA partition behavior. Protein partition towards the top phase was favored on systems constructed using PEG 1000 g/mol and TLL 45% (w/w), achieving up to 85% recovery. On the other hand a recovery of 92% was achieved at the bottom phase when PEG 3350 g/mol and TLL 25% (w/w) were used. Human serum was used as a complex sample on ATPS experiments. Selective fractionation of human serum proteins on ATPS constructed on flexible devices was achieved. ATPS constructed on blood bags required short equilibrium times (< 6 min), meaning it is feasible to use this approach on mass scale. The potential use of flexible disposable devices, for the fractionation of biological products using ATPS polymer–salt was demonstrated.     

Purification of the ADP-ribosylating enzyme from S. solfataricus by SDS-polyacrylamide gel electrophoresis and electroelution

The ADPribosylating enzyme from the thermophilic archaeon S. solfataricus was purified by a simple procedure which included preparative electrophoresis on a 0.1% SDS- polyacrylamide gel. The gel slice containing the enzymatic protein was cut out and the enzyme was solubilized by electroelution. The pure enzyme was obtained by chromatography of the electroeluted sample on a DNA-Sepharose column. The purified enzyme retained both its full activity and the structuring ability as a function of temperature increase.     

Recovery of DNA from agarose gels using a modified Elutrap.

We have made a significant improvement in the electroelution device, Elutrap (Schleicher and Schuell) by substituting an agarose gel barrier, which is made from 0.6% agarose (SeaKem GTG; FMC Corporation), into the elution chamber in place of the manufacturer specified BT2 membrane. This modification substantially increases the DNA recovery from agarose gels, even in samples containing less than 1 microgram of DNA, and shortens elution times particularly for large sizes of DNA (greater than 4.4 kbp). Additionally, the gel barrier provides a reproducible quantity and quality of DNA recovery. The high quality of the eluted DNA using the modified Elutrap makes this system suitable for further DNA manipulations.     

High-pressure gel loader for capillary array electrophoresis microchannel plates.

Microfabricated capillary array electrophoresis (microCAE) microchannel plates are the next generation of bioanalytical separation devices. To fully exploit the capabilities of microCAE devices, supporting technology such as robotic sample loading, gel loading, microplate washing, and data analysis must be developed. Here, we describe a device for loading gel into radial capillary array electrophoresis microplates and for plate washing and drying. The microplates are locked into a loading module, and high-pressure helium is used to drive aqueous separation media or wash solutions into the microchannels through fixtures connected to the central anode reservoir. Microplates are rapidly (30 s to 5 min) loaded with separation media, such as 3%-4.8% linear polyacrylamide or 0.7%-3.0% hydroxyethyl cellulose, for electrophoresis. The effective and rapid gel-filling and plate-cleaning methods together with short electrophoretic analysis times (2-30 min) make microCAE systems versatile and powerful nucleic acid analysis platforms.     

Advanced negative detection method comparable to silver stain for SDS-PAGE separated proteins detection

In order to achieve an easy, rapid and sensitive protocol to detect proteins in polyacrylamide gel, an advanced negative detection method comparable to silver stain is described. When a gel was incubated with Phloxine B and followed by the development in acidic solution, the zones where forming protein-dye complex were selectively transparent, unlike opaque gel background. Within 50 min after electrophoresis, down to 0.1–0.4 ng of gel-separated proteins (similar with silver stain) could be observed, without labor-intensive and time-consuming procedure. Comparing with the most common negative stain method, Imidazole-zinc stain, Phloxine B stain has been shown higher sensitivity and distinct contrast between the transparent protein bands/spots and opaque background than those; furthermore, it is no longer necessary to concern about retention time of observation. This technique may provide a sensitive and practical choice for proteomics researches.     

Electroelution of fixed and stained membrane proteins from preparative sodium dodecyl sulfate-polyacrylamide gels into a membrane trap

The membrane trap is a new device for the electroelution of all kinds of charged macromolecules from gels. Instead of dialysis membranes, the membrane trap uses a new membrane. Retention of macromolecules in an electric field by dialysis membranes depends on the presence of sodium dodecyl sulfate (SDS) in the buffer. The new membrane retains all charged macromolecules larger than approximately 5000 Da without adsorbing them, independent of the use of SDS. Here we report the electroelution of five different lipophilic membrane proteins (33 to 193 kDa) of Mycoplasma pneumoniae from preparative SDS-polyacrylamide gels into a 300-microliter recovery volume. After an 8-h elution period, recovery ranged from 80 (193 kDa) to 97% (33 kDa). The "losses" were generally due to proteins still remaining in the gel slice. All of the eluted proteins tested in a dot-blot assay proved to be antigenically active. The advantages of the device described here are easy handling (insertion of membranes, open system), quantitative recovery, and high reproducibility of the elution results.     

On-chip identification and interaction analysis of gel-resolved proteins using a diamond-like carbon-coated plate

We developed a novel protein chip made of a diamond-like, carbon-coated stainless steel plate (DLC plate), the surface of which is chemically modified with N-hydroxysuccinimide ester. To produce a high-density protein chip using the DLC plate, proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis were electroblotted onto the DLC plate and immobilized covalently. A high blotting efficiency (25-70%) for transferring proteins from the gels onto the DLC plates was achieved by improvement of the electrophoresis device and electroblotting techniques. With the use of the DLC plate, we developed novel techniques to identify proteins immobilized on the chip and to detect protein-protein interactions on the chip by mass spectrometric analysis. We also developed a technique to identify post-translationally modified proteins, such as glycoproteins, on the protein chip.