Monitoring of single nicks in duplex DNA by gel electrophoretic mobility-shift assay

We demonstrate that the gel electrophoretic mobility-shift assay (EMSA) can be used for site-selective and quantitative monitoring of nicks in linear double-stranded DNA (dsDNA) thus allowing to expediently follow the nicking activity of enzymes or other agents targeted to a designated dsDNA site. At elevated temperature and/or in the presence of urea, DNA fragments carrying a single nick produced by the nicking enzyme N.BstNBI exhibit a well-detectable gel retardation effect. On the basis of permutation analysis, the decreased electrophoretic mobility of nicked dsDNA fragments is attributed to a bend (or hinge) in the DNA double helix sequence-specifically generated by a nick. Since nick-induced DNA bending depends on interaction between base pairs adjacent to a nick, the change in mobility is different for nicked DNA sites with different sequences. Therefore, EMSA monitoring of differential mobility change caused by nicks within various DNA sequences could be useful for studying the differential base stacking and nearest-neighbor energetics.     

Blotting from PhastGel media after horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis

An improved procedure, "thermoblotting", is described for transferring proteins by diffusion from PhastGel Gradient media to an immobilizing matrix after horizontal sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After electrophoresis the gels were left on the separation bed of PhastSystem, the blotting matrix was applied and a transfer temperature was selected between 5-70 degrees C. An experimental series at fixed diffusion times showed that the transfer yield was significantly increased with temperature. The evaluation was done visually after staining of the blots with colloidal gold. An evaluation study comparing nitrocellulose, nylon, and polyvinylidenedifluoride of different pore sizes is also reported. Finally, the transfer efficiencies for 125I-labelled bovine serum albumin and soybean trypsin inhibitor were estimated using four different blotting procedures: two diffusion blotting techniques and two electrophoretic blotting techniques (tank vs. semi-dry).     

Stabilizing labile DNA–protein complexes in polyacrylamide gels

The electrophoretic mobility‐shift assay (EMSA) is one of the most popular tools in molecular biology for measuring DNA–protein interactions. EMSA, as standardly practiced today, works well for complexes with association binding constants K_a>10⁹ M^?1 under normal conditions of salt and pH. Many DNA–protein complexes are not stable enough so that they dissociate while moving through the gel matrix giving smeared bands that are difficult to quantitate reliably. In this work we demonstrate that the addition of the osmolyte triethylene glycol to polyacrylamide gels dramatically stabilizes labile restriction endonuclease EcoRI complexes with nonspecific DNA sequences enabling quantitation of binding using EMSA. The significant improvement of the technique resulting from the addition of osmolytes to the gel matrix greatly extends the range of binding constants of protein–DNA complexes that can be investigated using this widely used assay. Extension of this approach to other techniques used for separating bound and free components such as gel chromatography and CE is straightforward.     

An improved scheme for the identification of antigens recognized by specific antibodies in two-dimensional gel electrophoresis and immunoblotting

This paper describes an improved scheme for the identification of antigens in crude extracts recognized by specific antibodies when analyzed by a combination of two-dimensional gel electrophoresis and immunoblotting. First, protein components in gels are electrophoretically transferred to a polyvinylidene difluoride membrane which does not shrink or change dimensions in organic solvents. The efficiency of transfer and the localization of sample proteins on the membrane are checked and recorded by staining the blotting membrane with Fast Green FCF and recording the profile on a transparency. After blocking and the immunoassay, the results are recorded by photography. The sites of immune reaction are marked and the same membrane is restained briefly with Coomassie Brilliant Blue R-250 for the protein profile. Thus antigens in complex mixtures, recognized by antibodies of interest, can easily be identified from the restained membrane. If the whole protein profile is not well demonstrated, when used in combination with the profile recorded on the transparency, spots appearing on the restained membrane can still be used as useful landmarks in the final unequivocal antigenic identification. This improved scheme circumvents problems arising from membrane shrinkage and difficulties in accurately matching immunoreactive spots by conventional procedures and thus provides an accurate, simple and fast approach in the identification of antigens after immunoblotting.     

Native protein blotting after isoelectric focusing in fabric reinforced polyacrylamide gels in carrier ampholyte generated or immobilized pH gradients

An optimized procedure for the preparation of fabric reinforced polyacrylamide gels for native protein blotting is described. The gels, typically 5% T, 3% C, were internally stabilized with the aid of an AcrylAide-pretreated, hydrophilized polyester fabric, preferably with a 60 microns mesh opening. Ultrathin (120-180 microns) gels were prepared with the flap technique and 500 microns gels with the cassette technique; 500 microns gels with immobilized pH gradients were cast using precision molds and a computer controlled mixing device of four burettes. The fabric reinforced gels could be used either wet or after drying and rehydration. Isoelectric focusing was performed in carrier ampholyte pH gradients or hybrid immobilized pH gradients, supplemented with 1-3% w/v carrier ampholytes. Incorporation of 40-60% w/v glycerol into the gels decisively improved their operational properties. The high glycerol gels, which tolerated field strengths of 900-1700 V/cm for extended periods under steady state focusing conditions, were not afflicted by liquid exudation on the gel surface and showed retarded diffusion of the separated proteins on termination of focusing. By unidirectional capillary blotting, with an intermediate dialysis membrane eliminating bidirectional protein transfer, proteins were blotted to 0.1-0.2 micron pore size nitrocellulose membranes in 10-20 min from ultrathin gels and in 30-60 min from 500 microns gels. Based on quantification of residual protein in the gels after blotting, a transfer efficiency of 60-87% was found for the ultrathin and 53-69% for the 500 microns gels. Semidry electrophoretic blotting was carried out in a modified setup with cooled graphite electrodes. In a continuous Tris-glycine buffer system electrophoretic blotting required only 2-5 min with ultrathin gels and 20 min with 500 microns gels. Marker proteins, including horse spleen ferritin (Mr465,000), could be transferred with 91-96% efficiency.     

Method for detecting the lectin activity of Momordica charantia transferred from micro two-dimensional electrophoretic gel on to nitrocellulose.

A method was devised for detecting both the molecular mass and the isoelectric point (pI) of the lectin in the seed extract of Momordica charantia on a nitrocellulose membrane. It was associated with the electrophoretic blotting technique that produced replicas of proteins separated on micro two-dimensional polyacrylamide gels. The red blood cell adherence procedure on the blotted membrane exhibited only one red spot with molecular mass 107.10(3) and pI 5.3, which indicated the lectin activity. Additionally, the lectin appeared to be a glycoprotein with mannose and/or glucose, because it was stained by concanavalin A-peroxidase staining.     

Vertical agarose gel electrophoresis and electroblotting of high-molecular-weight proteins

The electrophoretic separation of high-molecular-weight proteins (> 500 kDa) using polyacrylamide is difficult because gels with a large enough pore size for adequate protein mobility are mechanically unstable. A 1% vertical sodium dodecyl sulfate (SDS)-agarose gel electrophoresis (VAGE) system has been developed that allows titin (a protein with the largest known SDS subunit size of 3000-4000 kDa) to migrate over 10 cm in a approximately 13 cm resolving gel. Such migration gives clear and reproducible separation of titin isoforms. Proteins ranging in size from myosin heavy chain ( approximately 220 kDa) up to titin can be resolved on this gel system. Electroblotting of these very large proteins was nearly 100% efficient. This VAGE system has revealed two titin size variants in rabbit psoas muscle, two N2BA bands in rabbit cardiac muscle, and species differences between titins from rat and rabbit muscle. Agarose electrophoresis should be the method of choice for separation and blotting of proteins with very large subunit sizes.     

Electroelution of nonfluorescent stacked proteins detected by fluorescence optics from gel electrophoretic bands for transfer into mass spectrometry

The extreme accuracy of spectrometrically determined masses of proteins has opened the possibility to identify proteins separated as gel electrophoretic bands in the absence of specific immunologic ways of identification. For the purpose of protein transfer from gel electrophoretic bands to mass spectrometer, electroelution from the intact gel has advantages, in particular when apparatus with capacity for fluorescent scanning allows one to direct the electroelution cell over the band under computer control. To avoid fluorescent labeling of the protein which is incompatible with mass spectrometric identification, it is proposed to selectively stack the unlabeled protein and detect it by comigrating tracking dye prior to electroelution. The feasibility of the approach is exemplified in case of a single protein, but still remains to be demonstrated in conjunction with the selective stacking or unstacking of a single protein from a mixture of several proteins.     

Important parameters in semi-dry electrophoretic transfer.

The efficiency of semi-dry electrophoretic transfer after sodium dodecyl sulfate (SDS)-electrophoresis using PhastGel media was investigated in a model system using three isotope labelled proteins. To give a full picture of the blotting process the amount of protein present in the gel, membranes, and filter papers was determined after different transfer times. The influence of the transfer buffer, commonly used additives such as methanol and SDS, and several different immobilizing matrices was investigated. Soybean trypsin inhibitor, bovine serum albumin, and ferritin were used as model proteins to study the effect of size on transfer efficiency. Basically, all three stages of the blotting process decide the result; the elution of protein from the gel, the immobilization of protein to the membrane, and the loss of material from the membrane during transfer. A theoretical explanation for the observed poor binding to a second membrane is discussed. Our results show that the buffer composition has little influence on the efficiency of transfer from the gel, but can be significant to the binding capacity of the membrane. In all experiments performed, there was never one moment during the transfer when all protein was eluted from the gel and simultaneously still bound to the membrane. The highest recovery in the membrane was obtained at different time intervals for different proteins. This indicates that quantitative transfer procedures cannot be generalized. However, obtaining an optimal method for reliable quantification of a specific protein or group of proteins is possible. For general protein staining of nitrocellulose and polyvinylidene difluoride membranes, a highly sensitive silver staining method requiring only 15 min has been used.     

The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins

The Pharmacia PhastSystem equipment has been used for crossed immunoelectrophoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second-dimensional electrophoresis, and only one overnight incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second-dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1% Triton X-100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.     

Sodium dodecyl sulfate free gel electrophoresis/electroelution sorting for peptide fractionation

Shotgun proteomics based on peptide fractionation by using liquid chromatography has become the common procedure for proteomic studies, although in the very beginning of the field, protein separation by using electrophoresis was the main tool. Nonetheless, during the last two decades, the electrophoretic techniques for peptide mixtures fractionation have evolved as a result of relevant technological improvements. We also proposed the combination of sodium dodecyl sulfate polyacrylamide gel electrophoresis for protein fractionation and sodium dodecyl sulfate free polyacrylamide gel electrophoresis for peptide separation as a novel procedure for proteomic studies. Here, we present an optimized device for sodium dodecyl sulfate free polyacrylamide gel electrophoresis improving peptide recoveries respect to the established electrophoretic technique off gel electrophoresis meanwhile conserving the excellent resolution described for the former technique in slab gel based systems. The device simultaneously allows the separation and the collection of fractionated peptides in solution.     

Multistrip Western blotting to increase quantitative data output

The qualitative and quantitative measurements of protein abundance and protein modification states are essential in understanding their role in diverse cellular processes. Traditional Western blotting technique, though sensitive, is prone to produce substantial errors and is not readily adapted to high-throughput technologies. We propose a modified immunoblotting procedure, which is based on simultaneous transfer of proteins from multiple gel-strips onto the same membrane, and is compatible with any conventional gel electrophoresis system. As a result, the data output per single blotting cycle can readily be increased up to ten-fold. In contrast to the traditional "one protein detection per electrophoresis cycle", this procedure allows simultaneous monitoring of up to nine different proteins. In addition to maintaining the ability to detect picogram quantities of protein, the modified system substantially improves data accuracy by reducing signal errors by two-fold. Multistrip Western blotting procedure allows making statistically reliable side-by-side comparisons of different or repeated sets of data. Compared to the traditional methods, this approach provides a more economical, reproducible, and effective procedure, facilitating the generation of large amounts of high-quality quantifiable data.     

Membrane-assisted online renaturation for automated microfluidic lectin blotting

Aberrant glycosylation plays a pivotal role in a diverse set of diseases, including cancer. A microfluidic lectin blotting platform is introduced to enable and expedite the identification of protein glycosylation based on protein size and affinity for specific lectins. The integrated multistage assay eliminates manual intervention steps required for slab-gel lectin blotting, increases total assay throughput, limits reagent and sample consumption, and is completed using one instrument. The assay comprises non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by online post-sizing SDS filtration and lectin-based affinity blotting. Important functionality is conferred through both device and assay advances that enable integration of nanoporous membranes flanking a central microchamber to create sub-nanoliter volume compartments that trap SDS-protein complexes and allow electrophoretic SDS removal with buffer exchange. Recapitulation of protein binding for lectin was optimized through quantitative assessment of SDS-treated green fluorescent protein. Immunoglobulin A1 aberrantly glycosylated with galactose-deficient O-glycans was probed in ~6 min using ~3 μL of sample. This new microfluidic lectin blotting platform provides a rapid and automated assay for the assessment of aberrant glycosylation.     

Improvement of electrophoretic transfer by casting acrylamide gels on a cellophane sheet

Electrophoretic transfer of protein after isolectric focusing using a polyacrylamide gel of less than 0.5 mm is difficult if the gel is backed to an electrically nonconductive casting support, such as glass plate or plastic films. By casting the gel on a cellophane sheet, it is not necessary to remove the gel from the support prior to electrophoretic transfer. The use of a cellophane support does not alter the quality of the final pattern.     

Modified diffusion blotting for rapid and efficient protein transfer with PhastSystem

An easy-to-perform and efficient blotting method was developed for the transfer of proteins separated in PhastGel media. The method was evaluated by comparing the blotting results of low molecular weight marker proteins at different times. 14C-Labelled proteins were used for the assessment of the transfer efficiency. Highly efficient transfer was achieved within 1 h of blotting and the proteins were almost completely eluted from the gel in 2 h. The gel remained on its solid plastic backing, which is not the case when using an electrophoretic transfer method. As little as 1.25 ng of electrophoretically separated protein were enzymatically detected on the blot when applying blot-biotinylation. The transfer was performed at low temperature (4 degrees C), which-in contrast to thermoblotting-additionally offers the chance to blot thermolabile proteins. These superior features, together with the method's versatility and low cost, mark an interesting alternative to the previously recommended blotting procedures after PhastSystem electrophoresis.     

Quantitative distance information on protein-DNA complexes determined in polyacrylamide gels by fluorescence resonance energy transfer

In polyacrylamide gels, we have quantitatively determined Forster transfer (fluorescense resonance energy transfer, FRET) between two fluorescent dyes attached to DNA in protein-DNA complexes. The donor-dye fluorescein labeled to DNA retains its free mobility in the polyacrylamide gel, however, its fluorescence properties change. The different quantum yield of fluorescein in the gel is found to be independent of the gel concentration and can thus be quantitatively taken into account by a reduced Forster distance R0 of 46 A compared to 50 A in solution. We have determined global structural properties of two proteins binding to double-labeled DNA using a novel gel-based fluorescence resonance energy transfer assay. In polyacrylamide gels we have analyzed the binding of integration host factor (IHF) and the high mobility group protein NHP6a to their substrate DNA. The measured Forster transfer efficiency allows us to deduce information on the overall shape and the DNA bending angle in the complex.     

A simplified method of protein electroblotting after isoelectric focusing on an ultrathin polyacrylamide gel layer fixed on a cellophane support.

A method is described for isoelectric focusing of proteins, using an ultrathin-layer polyacrylamide gel on cellophane, followed by electrophoretic transfer of separated proteins onto a nitrocellulose membrane. The polyacrylamide gel is firmly attached to the cellophane and thus protected from mechanical damage; such gels are easily manipulated. Cellophane is permeable to ions and application of this gel support overcomes difficulties resulting from the removal of ultrathin gels from a plastic support on electroblotting. Proteins separated under nondenaturing conditions were transferred onto a nitrocellulose membrane and detected by the concanavalin A-peroxidase technique. The proposed approach makes it possible to analyze the variability of nondenatured proteins and glycoproteins of different origin.     

A new isoelectric focusing system for fast two-dimensional gel electrophoresis using a low-concentration polyacrylamide gel supported by a loose multifilament string.

A new isoelectric focusing (IEF) system for two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been proposed. In this system, a super-soft and tough IEF gel was achieved by casting polyacrylamide gel down to 2.0% T using a loose multifilament string (LMS) of nylon as a gel support. The IEF apparatus for the LMS-gel, fabricated from acrylic boards, had a cooling water chamber, and eliminated the need of electrode solutions by directly connecting the two ends of individual gels to platinum electrodes. The carrier ampholyte-generated pH gradients using the new IEF system was stable over a long duration of time and a wide range of voltages, and the IEF time became shorter using a 2.0% T gel than using a 4.0% T gel. Also, the LMS-gels prepared in different runs exhibited excellent reproducibility. The new IEF system was applied to 2-D PAGE of a chicken skeletal muscle extract, and it was found that the protein loading capacity, protein entry into the LMS-gels, and protein transfer efficiency from the first-dimensional to the second-dimensional gels were significantly improved by using a low-concentration (2.5% T) gel. Also, proteins of high molecular weight of more than 200 kDa were observed in the 2-D maps, and therefore the new IEF system has a very good potential to be applied for fast 2-D PAGE of high molecular-weight proteins.     

Direct chemiluminescent immunodetection of proteins in agarose gels

Chemiluminescent immunodetection of proteins separated by polyacrylamide gel electrophoresis is generally performed only after Western blotting. Agarose gels are adequately permeable to allow immunoprobing directly in the gel. Chemiluminescent substrates had not been applied for direct immunoprobing of agarose gels. In a comparison with direct immunostaining of fibrinogen derivatives with horse radish peroxidase (HRP)-conjugated primary antibody using 3,3'-diaminobenzidene (DAB) yielding a sensitivity in the low nanogram range, a luminol-based chemiluminescent detection extended sensitivity to the mid-picogram range with seemingly no interference from either regular or glyoxyl agarose gels. The high sensitivity of chemiluminescence extends utility of direct immunoprobing of either agarose or glyoxyl agarose composite gels for detection and measurement of both high and low molecular weight proteins/peptides which are not easily detected/measured by Western blotting. However, due to the thickness of the gels, direct immunoprobing can be quite laborious. To eliminate that drawback, we describe a simplified approach, converting the thick gels to thin ones prior to probing, that makes direct immunoprobing as easy as Western blotting.