A Quantitative Procedure for Estimating Cotton Fiber Growth

An improved procedure for quantitation of cotton fiber development, the “stain-destain” method, is reported. Toluidine blue 0 was used to selectively stain fibers subsequently destained in an acid-alcohol solution. Absorbance of the dyecontaining destaining solution was used as a measure of fiber development, and expressed in terms of total fiber units (TFU), one OD unit at 624 nm having been assigned the value of one TFU. Optimum conditions for the procedure, including staining and destaining times and solution to ovule ratios were determined: (1) 20 ovules with associated fibers stained for 15 sec in 80 ml 0.018% toluidine blue O, (2) nonabsorbed dye removed by 60 sec wash, (3) ovuls destajned in 100 ml glacial acetic acid-ethanol-water (10:95:5), (4) absorbance determined after one hr destaining. The procedure is deemed accurate and precise for the purpose intended—quadtation of fiber development as modified by phytohormones or other treatments. Data are shown correlating TFU with fiber length through 14 days postanthesis and an example is given in which the method was used to determine the effect of combined application of gibberellic acid and indoleacetic acid on in vitro cotton fiber development.     

Polyacrylamide gel staining with Fe2+-bathophenanthroline sulfonate.

The utilization of Fe²⁺-bathophenanthroline sulfonate for the detection and quantitation of protein bands in cylindrical polyacrylamide gels is described. Two procedures are outlined. The first procedure is used in standard disc electrophoresis and involves fixing the protein with trichloroacetic acid, staining with Fe²⁺-bathophenanthroline sulfonate, and destaining with an ethanol:acetic acid solution. The second protocol reported is utilized with sodium dodecyl sulfate-containing gels. After electrophoresis, the gels are incubated with a methanol: acetic acid solution to remove the sodium dodecyl sulfate. The gels are then stained with Fe²⁺-bathophenanthroline sulfonate and destained with a methanol: acetic acid solution. Excellent background clarity is observed with both methods. Densitometric areas of the stained protein bands are linear to 60 μg of bovine serum albumin, and the limit of detection of this protein is 1 μg. Because of its rapidity of staining and destaining, good sensitivity, and reproducibility of stain intensity, Fe²⁺-bathophenanthroline sulfonate is an excellent protein stain.     

Gel gradient electrophoresis, isoelectric focusing and two-dimensional techniques in horizontal, ultrathin polyacrylamide layers

An ultrathin layer, horizontal polyacrylamide gel system for electrophoresis, isoelectric focusing and two-dimensional techniques is described. Gel slabs 240 micron thin for unidimensional, or 360 micron thin for two-dimensional runs are cast on cellophane foils as support. The sample is loaded in pockets pre-cast in the gel (2--3 microliter size) or in trenches for two-dimensional experiments. The second dimension is routinely performed in concave exponential gel gradients, spanning an acrylamide concentration from 4% to 22.5%. The sensitivity with the common Coomassie Blue stain is very high, well below 0.1 microgram protein/band. Zymogram detections can be developed within a few minutes, thus retaining the band sharpness of the focused zones or of the bands separated in pore gradient electrophoresis. Sample handling, staining and destaining and gel drying and storage are greatly simplified and performed in a fraction of the time needed for conventional, thick gels in the 1-2 mm thickness range.     

Heat mediated quick Coomassie blue protein staining and destaining of SDS-PAGE gels.

For the detection of proteins on sodium dodecyl sulphate-polyacrylamide gel electrophoresis, Coomassie blue is used commonly on account of its simplicity and reliability. In this report we show that enhanced heat, in addition to dramatically decreasing the time required for staining and destaining, also significantly increased the detection sensitivity. For a 1.5 mm gel, the staining time was 5 min at 55, 62.5 or 70 degrees C while the destaining time was 45, 45 and 20 min respectively. For a 0.8 mm gel, the staining time could be reduced to 1 min at 65 degrees C compared to 2 min at 60 degrees C and 5 min at 55 degrees C. The destaining time required was 8, 15 and 20 min at the respective temperatures.     

Two-dimensional polyacrylamide gel electrophoresis: an improved method for ribosomal proteins

A two-dimensional electrophoresis system for analysis of ribosomal proteins with several advantages over previous systems is described. The general features of this system are: (1) first-dimension separation on the basis of mobility at pH 5.0 in 8 m urea and 4% polyacrylamide; (2) second-dimension separation on the basis of molecular weight using dodecyl sulfate detergent; (3) rapid electrophoretic shift between first- and second-dimension separation conditions; (4) high resolution separation can be obtained on 10-cm² slabs with proteins from approximately 100 μg of ribosomal subunits; (5) capacity for handling up to 10 samples at a time, with electrophoresis complete within about 10 hr; and (6) the apparatus is relatively simple and inexpensive to construct and use.     

Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels

We present a new method for visualizing proteins electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are incubated in CuCl2 to produce a negative image of colorless protein bands against a semiopaque background. Gels are stained completely within 5 min, do not require destaining, and can be stored indefinitely without loss of the image. Because proteins are not permanently fixed within the gel, they can be quantitatively eluted after chelation of Cu with EDTA. The sensitivity of the CuCl2 stain falls between that of Coomassie blue and silver. We anticipate that CuCl2 will be useful in the rapid analysis of proteins by polyacrylamide gel electrophoresis and in the preparation of purified polypeptides by elution from gel slices.     

Detection of protein in polyacrylamide gels using an improved silver stain

A much improved silver staining procedure for the detection of protein in polyacrylamide gels of 0.8-3.0 mm thickness is described. It achieves very high sensitivity (detecting less than 0.01 ng bovine serum albumin/mm2) by overstaining and subsequently removing nonspecific background stain using a modified, reliable destaining procedure. Maximum sensitivity follows prediamine equilibration in 0.1% (w/v) formaldehyde solution. With two-dimensional electrophoresis the improved staining procedure reveals greater than 200 polypeptides in unconcentrated human urine and greater than 150 polypeptides in a single human fingerprint.     

A colloidal silver staining--destaining method for precise assignment of immunoreactive spots in two-dimensional protein patterns

The characterization of protein expression patterns by two-dimensional gel electrophoresis depends on efficient and reliable identification strategies for target spots. In addition to sophisticated techniques, such as microsequencing and peptide mass spectrometry, immunodetection of membrane-immobilized proteins is a valuable method with which to identify the corresponding spots for a given set of candidate proteins. To precisely assign immunoreactive spots, this approach requires specific immunodetection and staining of total protein to be performed on the same membrane. Here, we describe a highly sensitive, colloidal silver-based method for the assignment of immunoreactive spots in two-dimensional protein patterns. This simple and rapid procedure involves a destaining step after staining of nitrocellulose-bound proteins with colloidal silver. We show that destaining of proteins is a prerequisite for subsequent immunodetection using enhanced chemiluminescence. Several types of antibodies were successfully employed for antigen detection after the staining-destaining procedure. Our results demonstrate that the colloidal silver-based method is generally applicable for the unambiguous identification of candidate proteins in complex two-dimensional patterns.     

Micro-scale two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins

Summary A specially designed apparatus and conditions are described for the rapid analysis of ribosomal proteins by two-dimensional gel electrophoresis on a micro scale. The resolution of proteins in electropherograms is comparable to that obtained with other systems, but because of miniaturization, only 0.5 to 1 g of each protein is required, and the entire procedure, including electrophoresis in both dimensions, and staining and destaining can be completed in 6 to 7 hours.     

59Fe]Ferrous bathophenanthroline sulfonate: a radioactive stain for labeling proteins in situ in polyacrylamide gels

The use of a protein stain, [⁵⁹Fe]ferrous bathophenanthroline, to radioactively label proteins in polyacrylamide gels after electrophoresis using simple staining and destaining procedures is described.     

Destaining of Coomassie Brilliant Blue R-250-stained polyacrylamide gels with fungal laccase

An enzyme-based method for destaining polyacrylamide gels stained with Coomassie Brilliant Blue R-250 is described. Distilled water supplemented with diluted fermentation broth of a laccase-producing white-rot fungus, Cerrena sp., was used for gel destaining, and a clear gel background was obtained in 2 h at 37 °C. Sensitivity of protein detection was 10 ng. The method did not require organic solvents or changing the destaining solution. Due to simultaneous gel destaining and dye decolorization, the colorless destaining solution can be disposed of directly. Laccase destaining of polyacrylamide gels was simple, efficient, and environmentally friendly.     

Fluorescamine as a reagent for location of proteins after electrophoresis in starch gel or on paper

Fluorescamine (Fluram, Roche) has been used as a reagent for locating proteins after electrophoresis in starch gel and on paper. Attractive features of the method are speed, sensitity, and no destaining of gel or paper is required. The latex protein hevein, which is particularly difficult to stain by conventional methods, was detected very readily in starch gel and on paper with Fluorescamine.     

Development of a simplified, economical polyacrylamide gel staining protocol for phosphoproteins

Pro-Q Diamond (Pro-Q DPS) is a commercially available stain that binds the phosphate moiety of phosphoproteins with high sensitivity and linearity. To conserve consumable costs we demonstrate that threefold diluted Pro-Q DPS offers the same sensitivity and linearity of signal to that obtained with undiluted Pro-Q DPS. The optimal conditions for Pro-Q DPS indicate that fixation, staining, and destaining of gels longer than 1 h, 2 h, and four 30-min incubations, respectively, are not required. The fixation and destaining solutions, but not the threefold diluted Pro-Q DPS, can be re-used without compromising the signal intensity or linear dynamic range. This modified protocol of Pro-Q DPS reduces the cost at least by fourfold, making the stain economically attractive for large-scale analysis of phosphoproteins.     

Separation of very large DNA molecules by gel electrophoresis.

Very large DNA molecules were separated by electrophoresis in horizontal slab gels of dilute agarose. Conditions of electrophoresis were developed using intact DNA molecules from the bacterial viruses lambda, T4 and G. Their DNAs have molecular weights (M) of 32 million, 120 million, and 500 million, respectively. Several electrophoresis conditions were found which give sufficiently high mobilities and large differences that these DNAs are separated in a short time. Electrophoresis in 0.1% agarose at 2.5 V/cm of gel length separates T4 and lambda DNAs by 2.0 cm, and G and T4 DNAs by 1.0 cm in only 10 hr. With some conditions DNA mobilities are directly proportional to log M for M values from 10 to 500 million. The procedures used will allow rapid molecular weight determination and separation of very large DNA molecules.     

Role of very slightly deleterious mutations in molecular evolution and polymorphism

Several models of multiple slightly deleterious alleles are reviewed and theoretical consequences of slightly negative selection are discussed in conjunction with evolution and variation at the molecular level. Facts are organized which may be satisfactorily explained by the hypothesis of very slightly deleterious mutations. They are: (1) There appears to be an upper limit in heterozygosity for protein loci as measured by electrophoresis. (2) The excess of rare alleles is more pronounced in Drosophila than in man. (3) Correlation of heterozygosities at a locus among sibling species of the Drosophila willistoni group is too high compared to what is expected by the strict neutral theory, while it is not so among human races and between man and chimpanzee. (4) The rate of protein divergence is exceptionally high in Hawaiian Drosophila.     

Rapid phytochrome regulation of wheat seedling extension: light pretreatment extends coupling time, increases response lag, and decreases light sensitivity

We describe methods for densitometry of electrophoretically separated proteins in 25-millimeter microslab gels. The methods are sufficiently sensitive to use with several individually excised plant cells, for which we describe an extraction procedure. In brief, submicrogram samples are excised from freeze-dried plant tissue. Extraction takes place under oil in microliter droplets of detergent. Proteins are separated by one-dimensional microelectrophoresis (HM Poehling, V Neuhoff 1980 Electrophoresis 1: 90-102) and then stained by a sensitive Coomassie procedure (V Neuhoff, R Stamm, E Hansjorg 1985 Electrophoresis 6: 427-448). The resulting profile is scanned by a computerized densitometer based on the Leitz Diavert MPV Microphotometer. Evaluations and typical data demonstrate the high performance of this system.     

A charcoal cartridge for the removal of anionic detergent and electrophoresis stains

Instructions are given for the construction of a charcoal-containing cartridge that allows the rapid recirculation through charcoal of any fluid in which the cartridge is submerged; recirculatory flow is achieved by magnetic stirring of the fluid by a stirring bar placed under the cartridge. The device is assembled from nylon mesh and conical sections cut from polypropylene beakers. The device can be used to accelerate the destaining of electrophoresis gels and to remove SDS (sodium lauryl sulfate) from SDS gels. The removal of SDS prior to staining is essential for the staining of SDS gels with Coomassie Brilliant Blue G-250.     

Differences in phosphorylation of the two large subunits of brine shrimp Na,K-ATPase

Analysis of purified Na,K-ATPase from brine shrimp nauplii revealed two molecular forms of the alpha subunit separable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [G.L. Peterson, R.D. Ewing, S.R. Hootman, and F.P. Conte (1978) J. Biol. Chem. 253:4762]. The molecular form with lower mobility is designated alpha 1 and the one with higher mobility, alpha 2, in a neutral or alkaline gel system. Differences in Na+-dependent, K+-sensitive phosphorylation of these two molecular forms have been investigated by directly measuring the radioactivity present in each phosphoprotein after separation of the two forms by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of Na+,Mg2+, and ATP, when the ATP concentration is above 1 microM, both alpha subunits are phosphorylated, although the phosphoprotein content of alpha 1 is considerably greater than that of alpha 2. Below 1 microM ATP, the phosphoprotein content of alpha 2 is even further reduced. These striking differences in phosphorylation at low ATP concentrations are not due to a greater instability of the alpha 2 phosphoprotein during the long electrophoresis times or during fixation, staining, and destaining. The proportion of total phosphoprotein content in alpha 2, as well as the relationship between phosphoprotein content and ATP concentration, is unchanged when the radioactive analysis is performed on frozen gels that have been electrophoresed for shorter times, even though the actual amount of phosphorylation is 15 times greater than with fixed gels. Since the concentration of alpha 1 and alpha 2 vary during development [G.L. Peterson, L. Churchill, J.A. Fisher, and L.E. Hokin (1982) J. Exp. Zool. 221:295], the differences in phosphorylation may be relevant to differences in Na,K-ATPase activity during different development stages.     

Conditions for reproducible detection of calmodulin and S100 beta in immunoblots

The conditions used in some immunoblot procedures can fail to detect calmodulin, S100 proteins, and other proteins with similar physical properties. We describe here some of the basis of this difficulty, and provide an immunoblot protocol that allows the rapid and reproducible detection of calmodulin and S100 beta in crude biological samples. These proteins are rapidly transferred from sodium dodecyl sulfate-polyacrylamide gels to membrane matrices, and retention on the matrix is enhanced by a glutaraldehyde fixation step. Either nitrocellulose or a positively charged membrane filter (ZetaProbe) can be used as the immobilizing matrix. By combining microslab gel electrophoresis, 30 min electrophoretic transfer, and glutaraldehyde fixation of nitrocellulose paper, an immunoblot analysis can be done in an 8-hr day.     

Characterization of the H- and L-subunit ratios of ferritins by sodium dodecyl sulfate-capillary gel electrophoresis

Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) was used to characterize the H- and L-subunit ratios of several mammalian ferritins and one bacterioferritin. Traditionally, SDS-PAGE has been used to characterize the H- and L-subunit ratios in ferritin; however, this technique is relatively slow and requires staining, destaining, and scanning before the data can be processed. In addition, the H- and L-subunits of ferritin are fairly close in molecular weight (approximately 21,000 and approximately 20,000, respectively) and are often difficult to resolve in SDS-PAGE slab gels. In contrast, SDS-CGE requires no staining or destaining procedures and the peak quantitation is superior to SDS-PAGE. SDS-CGE is effective in quickly resolving the H- and L-subunits of ferritins from horse spleen, human liver, recombinant human H and L homopolymers, and mixtures of the two- and the single-subunit of a bacterioferritin from Escherichia coli. The technique has also proven useful in assaying the quality of the protein sample from both commercial and recombinant sources. Significant amounts of low-molecular-weight degradation products were detected in all commercial sources of horse spleen ferritin. Most commercial horse spleen ferritins lacked intact H-subunits under denaturing conditions.