Protein Staining Method Research Articles

Distinct ryanodine- and inositol 1,4,5-trisphosphate-binding sites in hepatic microsomes

A light hepatic microsomal preparation was fractionated by sucrose-density centrifugation into one rough, one intermediate and two smooth fractions. The four fractions were characterized with respect to parameters relevant to Ca2+ sequestration. Ca2(+)-ATPase activity was similar in the rough, intermediate and smooth I fractions, but lower in the smooth II fraction. Ca2+ accumulation was the highest in the smooth I and intermediate fractions. On the other hand, Ca2+ efflux from the rough fraction was several-fold faster than from the smooth I fraction. All four subfractions exhibited specific binding sites for inositol 1,4,5-trisphosphate (IP3) and ryanodine; however, the receptors were especially enriched in the smooth I fraction. The total binding sites for ryanodine in that fraction exceeded the number of binding sites for IP3 by about 10-fold. The two receptors responded differently to pharmacological agents; caffeine and dantrolene strongly inhibited ryanodine binding but not IP3 binding, whereas heparin inhibited IP3 binding only. Thus the two receptors are distinct entities. The four fractions also showed distinct gel electrophoretic patterns. The use of two different SDS/polyacrylamide-gel gradients and two protein-staining methods revealed major differences in the distribution of the bands corresponding to Mr values of (x 10(-3) 380, 320, 260, 170, 90, 29 and 21. These proteins were enriched in the smooth fraction. The results indicate that the smooth I fraction might have special importance in stimulus-evoked Ca2(+)-release processes.

A method for detecting proteins immobilized on nitrocellulose membranes by in situ derivatization with fluorescein isothiocyanate

A method for the fluorescent staining of proteins on nitrocellulose filters is described. The single step procedure uses a 100 μg/ml solution of fluorescein isothiocyanate in sodium carbonate buffer, pH 9.5. The proteins are visible under uv light within 30 s and the staining reaction is virtually complete after 10 min. The method can detect a minimum of 50 ng protein/band providing a sensitivity similar to that obtained with anionic dye stains. The method is suitable for blots prepared from both isoelectric focusing gels and sodium dodecyl sulfate-polyacrylamide gels. The fluorescently labeled proteins can be probed using immunochemical techniques with the retention of fluorescence. The method can therefore be used to accurately locate antigens among a number of proteins and allows the sensitive and rapid detection of marker proteins directly on the blot.

Genetic polymorphisms of the Pmo1 and Pmo2 salivary proteins detected by the modified protein staining method.

Two polymorphic proteins, Pmo1 and Pmo2, were found in human parotid saliva by modifying the protein staining method of Sung & Smithies (1969). The inheritance of each polymorphism was controlled by a dominant allele at an autosomal locus. This hypothesis was supported by studies in 50 families including 103 children. The gene frequencies were Pmo1+ = 0.308, Pmo1- = 0.692, Pmo2+ = 0.026, Pmo2- = 0.974. The Pmo1 and Pmo2 proteins reacted immunologically with antisera prepared to salivary proline-rich proteins (Pr and Gl). The isoelectric point was in excess of 8.58. These results showed that the Pmo1 and Pmo2 proteins belong to the basic proline-rich proteins in human parotid saliva.

Ultrastructural distribution of sulfated glycosaminoglycans in epithelial-mesenchymal interface of developing rat tooth germs.

We investigated the ultrastructural distribution of sulfated glycosaminoglycans in the epithelial-mesenchymal interface of tooth germs by use of the high-iron diamine thiocarbohydrazide silver proteinate (HID-TCH-SP) staining and enzymatic digestion method. At an early stage in odontoblast differentiation, HID-TCH-SP stain deposits were sparsely distributed in the basement membrane and in the intercellular spaces. Subsequently, as formation of the initial predentin matrix began, HID-TCH-SP stain deposits were densely distributed in the interfibrillar spaces and the basement membrane. Testicular hyaluronidase digested most of those in the progenitor pre-dentin, whereas those in the region of basal lamina resisted enzymatic digestion. Testicular hyaluronidase-resistant HID-TCH-SP stain deposits were susceptible to heparitinase, indicating that the sulfated glycosaminoglycan in the basal lamina is heparan sulfate. Furthermore, the heparan sulfate tended to be regularly arranged at the sites of internal and external lamina densa. However, as progenitor pre-dentin matrix formation proceeded, the numbers of stain deposits temporarily increased and their distribution pattern became irregular, finally tending to disappear with the disruption of basal lamina.

A rapid method for staining proteins in acrylamide gels

A negative staining procedure for the rapid visualization of proteins in acrylamide gels is described. In the absence of proteins, staining of the gel occurs through the reduction of nitroblue tetrazolium by reduced glutathione. No staining occurs in the presence of proteins. The procedure can be completed in 20 min and is at least as sensitive as Coomassie brilliant blue staining.

FerriDye: Colloidal iron binding followed by Perls' reaction for the staining of proteins transferred from sodium dodecyl sulfate gels to nitrocellulose and positively charged nylon membranes

A staining method for proteins on (positively charged) nylon and nitrocellulose membranes is described. The two-step method uses cationic cacodylate iron colloid which is substituted with Tween 20 at an OD 460 nm = 0.5, followed by Perls' reaction with acid potassium ferrocyanide. It stains transferred proteins deep blue with low background. The sensitivity is intermediate between that of conventional stains and AuroDye, the colloidal gold stain. This is the first sensitive staining method for proteins transferred on (positively charged) nylon membranes. These membranes have documented advantages in immunoblotting. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Histochemical Protein Staining Methods

Histochemical Protein Staining Methods

Fluorescent staining of sialidases in polyacrylamide gel electrophoresis and ultrathin-layer isoelectric focusing

Polyacrylamide gels were stained with the sialidase substrate 2′-(4-methylumbelliferyl)-α- d- N-acetylneuraminic acid showing the activity of Vibrio cholerae and Clostridium sordellii sialidases in the gels after electrophoresis. With this fluorogenic method minimum sialidase activities of 5 μU could be determined. The sensitivity of this staining is about 10,000-fold higher compared to protein-staining with Coomassie brilliant blue. For the visualization of other proteins than sialidases the specific sialidase staining could be followed by a protein-staining method in the same gel.

A fluorescent method for the rapid staining and quantitation of proteins in sodium dodecyl sulfate‐polyacrylamide gels

AbstractFluorescence studies carried out in solution indicate that in the presence of the anionic detergent sodium dodecyl sulfate (SDS) proteins are able to bind the noncovalent dye 1‐anilinonaphthalene‐8‐sulfonate (ANS). The interaction of this hydrophobic dye with protein‐SDS complexes is probably responsible for the fluorescence of protein bands observed by UV‐transillumination of polyacrylamide gels stained with ANS. It is shown that the staining of SDS‐polyacrylamide gels with ANS after electrophoresis allows the detection and quantitative estimation of proteins and peptides having different properties. The electrophoretic patterns obtained using ANS are equivalent to those obtained using Coomassie Brilliant Blue R‐250. The fluorescent staining method described in this work is simpler and much more rapid than conventional methods using visible dyes.

Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining

AbstractThe study of effects of several parameters on silver staining of proteins has led to the development of a staining method which is simple and reliable, requires only few stable solutions, and can be applied to all gel types such as sodium dodecyl sulfate (SDS) containing polyacrylamide or isoelectric focusing gels. It can be used for ultrathin layers (0.1–0.2 mm) or thicker slab gels (up to 3 mm). With this method not only proteins but also polypeptides of molecular weights as low as 2500 are detectable with high sensitivity. Comparison of isoelectric focusing and SDS‐containing gels and a simple spot test on thin gellayers show that the detection sensitivity depends not so much on the type of proteins but rather on their structure. The redox properties of the gel are important for the staining mechanism.

Gel Electkophoretic Separation of Angiotensins I, II and III

Abstract The hepta-, octa- and decapeptides, angiotensins III, II, and I with molecular weights 930, 1,045, and 1,295, were separated by polycrylamide pel electrophoresis at pH 6.41, positive polarity, using gel concentrations ranging from 24 to 40 %T, 2 %CBis. The peptides were detected by a conventional protein staining method using Cooraassie Brilliant Blue R-250 modified by the addition of 1.25% glutaraldehyde, thus crosslinking the peptides to “proteins”. Separation between the 3 peptides Is due nearly entirely to net charge rather than to size. Resolution is therefore optimal in charge fractionatton methods such as electrofocusing, chromatofocusing, ion exchange chromatography and liquid-liquid partition chromatography.

Abnormal Distribution of Complex Carbohydrates in Neutrophils of a Patient With Lactoferrin Deficiency

Previous studies have identified patients with susceptibility to bacterial infection associated with lactoferrin deficiency in dysmorphic neutrophils containing abnormal or no secondary granules and abnormal nuclear segmentation. We have investigated the subcellular distribution of vicinal glycol-containing complex carbohydrates in marrow and blood myeloid cells of such a patient using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining method and have examined the response of these neutrophils to the degranulating agents N-formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). As in normal specimens, immature primary granules were strongly PA-TCH-SP reactive; however, unlike normal specimens, masking of PA-TCH-SP reactivity did not occur in mature primary granules. Endoplasmic reticulum demonstrated moderately strong PA-TCH-SP staining, in contrast to absent staining of this organelle in normal promyelocytes and consistent with abnormal primary granule genesis. Small abnormal elongated granules (0.1-0.2 micron in diameter) were identified at the myelocyte state of development and were the predominant granule type in late neutrophils. These granules were identified as secondary granules on the basis of their PA-TCH-SP positivity and were differentiated from primary and tertiary granules on the basis of a lack of peroxidase, acid phosphatase, and sulfate staining. When the neutrophils were exposed to PMA, cell aggregation occurred, and the abnormal granules degranulated in a manner similar to the degranulation observed with normal secondary granules. Although PA-TCH-SP staining of the plasma membrane appeared normal, a decrease in FMLP receptors was demonstrated. Thus, a defect(s) is present in complex carbohydrate distribution and staining that involves primary and secondary granules and possibly the plasmalemma of neutrophils from this patient. This results in abnormal packaging of primary granules and synthesis of normal numbers of secondary granules that are qualitatively and morphologically abnormal, but can be recruited to degranulate with PMA.

Abnormal distribution of complex carbohydrates in neutrophils of a patient with lactoferrin deficiency

Previous studies have identified patients with susceptibility to bacterial infection associated with lactoferrin deficiency in dysmorphic neutrophils containing abnormal or no secondary granules and abnormal nuclear segmentation. We have investigated the subcellular distribution of vicinal glycol-containing complex carbohydrates in marrow and blood myeloid cells of such a patient using the periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) staining method and have examined the response of these neutrophils to the degranulating agents N-formylmethionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA). As in normal specimens, immature primary granules were strongly PA-TCH-SP reactive; however, unlike normal specimens, masking of PA-TCH-SP reactivity did not occur in mature primary granules. Endoplasmic reticulum demonstrated moderately strong PA-TCH-SP staining, in contrast to absent staining of this organelle in normal promyelocytes and consistent with abnormal primary granule genesis. Small abnormal elongated granules (0.1–0.2 micron in diameter) were identified at the myelocyte state of development and were the predominant granule type in late neutrophils. These granules were identified as secondary granules on the basis of their PA-TCH-SP positivity and were differentiated from primary and tertiary granules on the basis of a lack of peroxidase, acid phosphatase, and sulfate staining. When the neutrophils were exposed to PMA, cell aggregation occurred, and the abnormal granules degranulated in a manner similar to the degranulation observed with normal secondary granules. Although PA- TCH-SP staining of the plasma membrane appeared normal, a decrease in FMLP receptors was demonstrated. Thus, a defect(s) is present in complex carbohydrate distribution and staining that involves primary and secondary granules and possibly the plasmalemma of neutrophils from this patient. This results in abnormal packaging of primary granules and synthesis of normal numbers of secondary granules that are qualitatively and morphologically abnormal, but can be recruited to degranulate with PMA.

Simultaneous staining of proteins during polyacrylamide gel electrophoresis in acidic gels by countermigration of Coomassie brilliant blue R-250

A method for the simultaneous staining of proteins during polyacrylamide gel electrophoresis with Coomassie brilliant blue R-250 at pH 2.5 is described. Calf thymus whole histone and cytochrome c were stained by this method and the results obtained were similar to that obtained by staining after electrophoresis.

Analysis of human palatine salivary proteins by isoelectric focusing in agarose

An electrophoretic method capable of analysing a small volume (5–10 μl) of human palatine saliva without prior concentration was developed by combining the the high resolution of isoelectric focusing in agarose with a highly sensitive silver protein staining method. Human palatine saliva exhibited 23 protein bands, those at pI 4.3–4.4, 4.7, 5.3 and 5.8 being major bands.

Rapid staining of proteins in ultrathin‐layer isoelectric focusing in polyacrylamide gels

AbstractA method for protein staining after isoelectric focusing (IEF) in polyacrylamide gel is described in which the total time for fixation, staining and complete gel background destaining is reduced to 3–5 min. This is achieved by (i) staining dry gels on silanized supports instead of hydrated gels, (ii) reducing gel thickness to 50 μm, (iii) using Servalyt carrier ampholytes with good destaining properties, and (iv) using dyes with low affinity for carrier ampholytes, namely Serva Violet 17 or 49, facilitating destaining of gel background. The sensitivity of staining is 100–200 ng protein/ mm2 over a 10 cm separation distance and 20–30 ng protein/ mm2 over 3 cm gels (miniature focusing). Addition of 5–20 % methyl acetate to the staining or destaining solution reduces the sensitivity of staining significantly. Most effective fixation of proteins in 50 μm gels is achieved with 20% trichloroacetic acid. In solutions of alcohol and acetic acid, appreciable amounts of proteins are leached after incubation for 5–20 min. Paraformaldehyde, malondialdehyde or glutaraldehyde (5–10 %) exhibit no fixative effect on proteins in 50 μm gels when used directly or after precipitation of the proteins with trichloroacetic acid. The selective leaching of proteins under a variety of experimental conditions shows that adequate fixation is particularly important in work with basic, low molecular weight and/or hydrophobic proteins.

Quantitative cytochemistry of nuclear and cytoplasmic proteins using the Naphthol Yellow S and dinitrofluorobenzene staining methods.

The 'total protein staining' of biological specimens with the electrostatically binding Naphthol Yellow S or the covalently binding dinitrofluorobenzene must be interpreted as methods which yield data on the specific amino acid pool of the proteins concerned. Both dyes bind to certain free amino-acid side-chains, giving different dye--protein ratios for various proteins. In the presence of DNA, dinitrofluorobenzene stains all proteins present in cell nuclei, whereas Naphthol Yellow S only stains the majority of the non-histone proteins. When protein staining methods are combined with the Feulgen--Pararosanile (SO2) procedure for DNA, decreased Feulgen--DNA contents were measured in dinitrofluorobenzene-stained isolated nuclei and lymphocytes.

Simplified method for DNA and protein staining of human hematopoietic cell samples.

A rapid reproducible method yielding high resolution analysis of DNA and protein in human hematopoietic cell samples has been developed by modification of the propidium iodide and fluorescein isothiocyanate procedure. Cell staining involves sequential addition of each reagent (RNase, fluorescein isothiocyanate and propidium iodide) to ethanol-fixed cells and requires no centrifugation steps. Stained cells are analyzed in the reagent solutions. Analysis of bone marrow samples from multiple myeloma patients showed mixed normal and aneuploid populations with a major portion of the aneuploid cells having a significantly higher protein content. This approach permitted differential cell cycle analysis of normal and the aneuploid populations.

Protein staining methods in quantitative cytochemistry.

The chemical action and practical application of the Naphthol Yellow S, Alkaline Fast Green, Coomassie Brilliant Blue, Dinitrofluorobenzene and some lesser known protein staining methods have been surveyed with respect to their potentialities for quantitative cytochemical analyses. None of the dyes can be said to bind to any specific protein or group of proteins, but each may be used to analyse the presence of one or more particular amino acid residues. For the cytophotometric measurement of the 'total protein content' of individual cells and cell organelles the covalent binding Dinitrofluorobenzene and the electrostatic binding Naphthol Yellow S can properly be used. Fast Green FCF, applied at alkaline pH, binds electrostatically to the basic amino acid side chains of strongly basic proteins only but not in a quantitative (stoichiometrical) way. Coomassie Brilliant Blue, recently introduced to protein cytochemistry, may be useful for quantitative purposes. The combined Feulgen-Pararosaniline(SO2)/Naphthol Yellow S and Dinitrofluorobenzene/Feulgen-Pararosaniline(SO2) methods enable the simultaneous cytophotometric analysis at two different wavelengths for protein and DNA within the same microscopical preparation.

Ultrastructural localization of polysaccharides in the vacuolar system of an established cell line

Polysaccharides in the vacuolar system of an established line of monkey kidney epithelial cells, were investigated by high resolution radioautography following incorporation of 3H-fucose and by the periodic acid-thiosemicarbazide-silver proteinate staining method. Significant amounts of glycogen and glycoproteins were found in different lysosomes and prelysosomes. The radioautographic labeling of these substances correlated with the cytochemical observations. Analysis of the 3H-fucose pattern of labeling suggests that glycoproteins are transported through the different components of the vacuolar system of the cell in a sequential fashion rather than via independent pathways. The possible functional significance of polysaccharides in lysosomes is discussed. It is suggested that glycogen is taken up by lysosomes through autophagic segregation and through infolding and vesiculation of the lysosomal surface, and that the majority of glycoprotein in the lysosomal membranes does not have acid hydrolase activity.