Two methods for electrophoretic elution of proteins from polyacrylamide gels.

An apparatus is described which improves an earlier technique for eluting proteins from polyacrylamide-gel slabs by electrophoresis against a sucrose gradient. Another elution method where the components are concentrated electrophoretically in a collodion bag by altering the current density is described. This method enables the elution of small amounts of sample, free from disturbing background material arising from the gel, and also permits subsequent dialysis and ultrafiltration without transfer losses. It can be used with alkaline and acidic buffers and has been applied in the purification of human pituitary thyrotropin (TSH).     

Electrophoretic elution of macromolecules from polyacrylamide or agarose gels

A method for elution of micrograms of macromolecules from polyacrylamide and agarose gels is described. The recoveries were greater than 90% with three different macromolecules tested (28 to 360 kDa). An amount as small as 1 microgram of human serum albumin was eluted from polyacrylamide gel with 90% recovery. The eluted material is collected into a small chamber the size of which can be changed as required. Elution and concentration are achieved simultaneously and in one step under mild conditions. Sterile eluates can be obtained, if the apparatus is constructed under sterile conditions.     

Visualization and elution of unstained proteins from polyacrylamide gels

Proteins fractionated by electrophoresis on 18% polyacrylamide gels with low crosslinking can be directly visualized by ultraviolet light-induced fluorescence and can be recovered by electroelution.     

Optimized centrifugation for rapid elution of DNA from agarose gels

A simple method has been developed for the isolation of DNA from agarose gels. Centrifugation in a low-cost device for 45 s at low speed provides a high yield of DNA suitable for further manipulation by restriction enzymes, T₄ DNA ligase, Taq polymerase, Klenow fragment, and T₄ polynucleotide kinase, and also for sequencing. In contrasr, centrifugation for > 1 min leads to coelution of substances that inhibit DNA ligase.     

Laser excitation of fluorescent-labeled polypeptides in polyacrylamide gels

A laser beam at 488 nm, converted into a fan of light by a surface-coated mirror oscillated in response to a triangular wave, was inserted into the base of a polyacrylamide gel. The laser light was trapped by internal reflection and gave uniform illumination throughout the entire gel slab. Photography with color film detected 50 fmol of fluorescein covalently coupled to ovalbumin, gave 80-fold greater sensitivity than transillumination in detection of fluorescein-labeled polypeptides, and was about 25-fold more sensitive than protein staining with silver. Laser illumination visualized end-labeled beta-galactosidase, afforded quality control of such preparations, and demonstrated that the end-labeled derivative contained about 25-fold less fluorescein than uniformly labeled beta-galactosidase. The latter result was confirmed by dot-blot analysis using a polyclonal antibody specific for fluorescein. The application of end-labeling to the location of features of protein primary structure is discussed.     

Functional renaturation of receptor polypeptides eluted from SDS polyacrylamide gels

In order to gain further support for the concept that a homo-oligomeric protein-complex may be sufficient to form a functional ligand-activated ion channel and to explore additional possibilities for the reconstitution of channel activity, a single polypeptide band of the purified neuronal AChR from insects has been electroeluted from SDS-polyacrylamide gels, the SDS removed and the polypeptides incorporated into liposomes. Liposomes were fused into planar lipid bilayers which were subsequently analysed for channel activity. Fluctuations of cation-channels were detected after addition of agonists (carbamylcholine); channel activity was blocked by antagonists (d-tubocurarine). The channels formed by electroeluted polypeptides gave conductance values, as well as kinetic data, quite similar to channels formed by the native receptor protein. Sedimentation experiments using sucrose density gradient centrifugation revealed that a considerable portion of the electroeluted polypeptides assembled during the reconstitution process to form oligomeric complexes with a sedimentation coefficient of about 10 S; thus resembling the native receptor complex. Offprint requests to: W. Hanke     

Recovery of biologically functional messenger RNA from agarose gels by passive elution

A general method for isolating biologically active messenger RNA (mRNA) from agarose gels is reported. Purified cellular RNA is resolved by preparative agarose gel electrophoresis and recovered in high yields (80%) by passive diffusion. Polyadenylated mRNA isolated from the eluted RNA is functionally intact based on the ability of the RNA to serve as a template in cell-free translation systems and complementary DNA synthesis reactions. The entire procedure is simple and rapid. A substantial purification of the mRNAs coding for skeletal muscle myosin heavy chain, light chain subunits and carbonic anhydrase III has been achieved employing this method.     

The isolation of DNA from agarose gels by electrophoretic elution onto malachite green-polyacrylamide columns

Electrophoretic elution of DNA coupled with direct adsorption onto malachite green-polyacrylamide columns was used to isolate double- and single-stranded DNA from agarose gels. Subsequently, DNA was eluted with a high salt buffer and filtered through Sephadex which permitted recovery of the DNA in a low salt buffer at concentrations suitable for heteroduplex analysis by electron microscopy. This method was tested by examining hetero-duplexes formed from the isolated complementary single strands of T7 wild type DNA and a T7 deletion mutant. More than 80% of the reannealed molecules were intact heteroduplexes showing the deletion loop. Irradiation of single-stranded DNA with 254 nm light resulted in distorted, convoluted heteroduplexes while 366 nm light did not show this effect.     

Interaction of TMV-proteins during electrophoretic separation in polyacrylamide gels

Summary The technique of polyacrylamide gel electrophoresis was applied to demonstrate interaction between proteins of different strains of tobacco mosaic virus. The advantages and limitations of the method for such studies have been indicated. The results are discussed in connection with the role of primary structure of TMV-proteins for mixed aggregations.The present observations were reported by us under the title, Mixed Aggregation of Protein Sub-Units of Tobacco Mosaic Virus Mutants: Role of Primary Structure, during a symposium on Interaction between Subunits of Biological Macromolecules, organized by the International Union of Pure and Applied Biophysics, and held in Cambridge, U.K., from 24–27th June, 1968.     

Chromatographic recovery of polypeptides from copper-stained sodium dodecyl sulfate polyacrylamide gels.

A procedure of preparative electrophoresis is described in which proteins separated on sodium dodecyl sulfate gels, stained with copper and eluted by simple diffusion, are highly concentrated on a fluorocarbon packing and freed of small molecular weight substances, including sodium dodecyl sulfate and buffer components and gel-related substances. This method can be used for microscale preparations or it can be scaled up to recover milligram amounts of protein. The purified polypeptides, however denatured, are suitable for amino acid sequencing.     

Advantages of picrate fixation for staining polypeptides in polyacrylamide gels

When acetic acid-urea polyacrylamide gels with or without Triton X-100 were immersed in 0.1 M Na picrate, pH 7, to which 1/4 vol Coomassie blue staining solution (0.2% in 45% methanol, 10% acetic acid, 45% water) was added, proteins stained rapidly (within a few minutes in gels without Triton and within an hour in gels with Triton) with little or no background staining. Thus protein bands could be observed in a single step with no destaining. The picrate-Coomassie blue method fixed and stained a small peptide (bradykinin, nine amino acids) that was not observed in gels stained with fast green, silver, or Coomassie blue following fixation in 50% trichloroacetic acid. The picrate-Coomassie blue method gave high-contrast bands suitable for densitometry. Gels containing sodium dodecyl sulfate were also stained by the picrate-Coomassie blue method if they were first washed briefly (1 h) in 45% methanol, 10% acetic acid, 45% water, presumably to remove the detergent. These gels also stained rapidly with almost no background.     

Quantitative electrophoretic transfer of DNA from polyacrylamide or agarose gels to nitrocellulose

A method for efficient electrophoretic transfer of DNA fragments from polyacrylamide gels to nitrocellulose sheets was developed. Hybridization to these fragments can be performed by standard techniques. The method is also applicable to agarose gels, allowing this transfer method to be used for DNA ranging from 40 to at least 23,000 bp.     

Quantitation of specific proteins in polyacrylamide gels by the elution of fast green FCF

The quantitation of proteins in polyacrylamide gels stained with Fast Green FCF has been investigated using a modification of the elution technique originally described by Fenner et al. (Fenner, C., Traut, R.R., Mason, D.T. and Wikman-Coffelt, J. (1975) Anal. Biochem. 63, 595–602) for Coomassie Blue and adapted by Medugorac (Medugorac, I. (1979) Basic Res. Cardiol. 74, 406–416) for use with proteins stained with Fast Green FCF. The elution of dye from stained protein was accomplished using 1.0 M NaOH instead of aquoeus pyridine as required by the original method. The primary advantages of our modification are that the time required for protein quantitation has been considerably reduced and the use of toxic organic solvents has been eliminated. We have investigated the applicability of the method to several different proteins and our results indicate: (a) The quantity of Fast Green eluted from specific proteins is proportional to the quantity of protein applied to the gel, but varies for each individual protein. (b) The method allows quantitation over a very wide range of protein (1–800 μg). (c) Quantitation of protein is independent of the width of the stained bands as well as acrylamide concentration. (d) The method is applicable to gels of many types including disc, slab and continuous gradient gel, (e) Protein can be estimated from the patterns obtained by two-dimensional polyacrylamide gel electrophoresis. (f) The presence of Triton X-100 in gel and protein sample does not affect quantitation; the method is applicable to gels containing SDS provided that SDS is removed prior to staining. (g) Precipitation of protein with 12.5% TCA following electrophoresis does not interfere with quantitation. (h) The reproducibility of the technique is excellent, with standard deviations being less than 10% of the mean in all cases. This method appears highly versatile but requires appropriate standards for the quantitation of individual proteins.     

A simple and fast electrophoretic method for elution of nucleic acids from gels

A very convenient electrophoretic procedure for DNA or RNA elution from agarose or polyacrylamide gels is described. The gel piece with nucleic acid to be eluted is contained in a dialysis bag filled with buffer and elution is carried out in a horizontal electrophoresis apparatus. The nucleic acid is recovered with a high yield and can be used, without prior treatment, in further enzymatic or chemical reactions. Results obtained with DNA are presented here.     

Charge-dependent staining of proteins after electrophoretic separation in polyacrylamide gels

A staining procedure is described which allows for the identification of basic and acidic proteins after gel electrophoresis. This includes electrophoresis of sodium dodecylsulfate (SDS) membrane proteins not accessible to isoelectric focusing as a means of charge-dependent separation. Nonfixative charge-dependent staining can be used for detection of proteins after separation in gels, when further investigation of the intact protein is desired.     

A rapid sensitive silver stain for polypeptides in polyacrylamide gels

The use of silver to detect polypeptides was originally achieved by modifying tissue stains. By adapting methods of photochemistry we have developed a new silver stain for polypeptides which is nearly as sensitive but much more efficient than these earlier procedures. The new silver stain utilizes only three solutions and allows protein patterns to be visualized within 50 min. Its sensitivity is 100 times that of the Coomassie blue stain.     

Electroblotting of individual polypeptides from SDS/polyacrylamide gels for direct sequence analysis

A scheme for electroblotting of individual unstained protein bands from SDS/polyacrylamide gels and subsequent amino acid sequence analysis is described. Principal features are: detection of the polypeptide bands by visualization with KCl; electroblotting of excised gel pieces that correspond to the protein bands only; blotting onto polybrene-pretreated glass-fiber filter discs (12 mm diameter) placed in an electrophoretic concentrator. A high yield over all steps from gel application through electrophoresis, blotting, gas-phase sequencer degradation, and phenylthiohydantoin analysis is obtained with several different types of polypeptide (combined average yield over all steps 20%, spread 10-50%). Background is low and samples can be stored under vacuum for long periods after blotting.     

A discontinuous electrophoretic system for separating peptides on polyacrylamide gels

An electrophoretic system for separating, with high resolution, peptides 25–250 residues in length is described. The peptides are stacked by discontinuous electrophoresis to form very sharp bands at the origin. They are then separated on a matrix of 20% polyacrylamide, 8 m urea, and 0.1% dodecyl sulfate. Through this combination, high resolution and clean separation, based on polymer length, are achieved.     

Genetic analysis of thirty-three platelet polypeptides detected in two-dimensional polyacrylamide gels.

Two-dimensional gel electrophoresis followed by silver-staining was utilized to visualize platelet polypeptides for genetic analysis. A subset of 33 polypeptides that were most suited for scoring was selected. Families consisting of father-mother-child trios were studied. Thirty-six polypeptides of a total of 1,413 scored in children's gels exhibited the combination of a normal and a variant polypeptide. The observed index of heterozygosity of 2.55% is comparable to our previously reported findings for red cell proteins.     

A study of electrophoretic mobility of DNA in agarose and polyacrylamide gels

The aim of this paper is to clarify the mechanism of gel electrophoresis of DNA under constant-field conditions. We have conducted a large number of experiments on double-stranded DNA varying in length between approximately 10 and approximately 50,000 base-pairs, in both agarose and polyacrylamide gels ranging from 0.5% to 12% concentration, and with electric field strengths ranging from 0.5 to 8 V/cm. We have made (logarithmic) plots of velocity against length of DNA for all of the various test conditions. At the left-hand side of these plots, all of the empirical curves have a unique, standard shape. When the curves are normalized so that their left-hand parts coincide, a second feature emerges in that, while for any given test the curve follows the "master curve" up to a certain point, it then "breaks away" and becomes horizontal. We describe these two patterns of behaviour as "regions 1 and 2", respectively. We find simple yet comprehensive empirical formulae that fit the observations in the two regions of behaviour: these express the velocity in terms of length of DNA, electric field strength and gel concentration. We then construct two separate theories for the two regions of behaviour. The first theory involves the statistics of motion of an object through a random array of gel obstacles, with the instantaneous speed depending on the number of obstacles with which the object is currently in contact. The second theory is based on the mechanical hypothesis (for which there is other, independent support) that the DNA moves through the gel by piling up against a barrier, which eventually breaks or deforms under the resulting force, thereby allowing the DNA to move on to the next barrier. The statistical theory is an adaptation of existing work, while the mechanical one is new. We also describe experiments on the migration of repeated-sequence, curved DNA with length up to 1500 base-pairs, and we discuss its behaviour in terms of our two theories. Our studies by electron microscopy are consistent with the view that this repeated-sequence DNA adopts a superhelical configuration. Finally, we show that a very wide range of observations may be understood clearly by means of our two theoretical schemes.