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PubMed1988

Isolation of proteins for peptide mapping, amino acid analyses, and sequencing using disulfide crosslinked polyacrylamide gels.

Conditions for recovery of small amounts of proteins (1-50 micrograms) from disulfide crosslinked polyacrylamide gels have been examined. Procedures were developed for solubilization and precipitation of Coomassie blue-stained protein bands excised from gels after electrophoretic separations. The precipitated protein was then resolubilized for use in peptide mapping, amino acid analyses, or microsequencing. The amino acid compositions of standard proteins (bovine albumin, ovalbumin, phosphorylase b, and beta-galactosidase) isolated by this method were in good agreement with the values for the corresponding conventionally purified proteins. Sequencing was done with high repetitive yield on samples of 100 pmol or below. The method has been successfully applied to several proteins and protein fragments.

Amino Acid Sequence↗
PubMed1988

Lysozyme oligomers as a molecular mass standard for sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Egg white lysozyme treated with hypochlorous acid links together producing di-, tri-, tetra-, and pentameric derivatives with molecular masses ranging from 14,300 to 90,500. Similar oligomeric products may be obtained by treating lysozyme color derivatives produced by labeling lysozyme with fluorescein, trinitrobenzenesulfonic acid and 2,4-dinitrofluorobenzene, with hypochlorous acid. The oligomeric lysozyme derivatives thus obtained consist of a mixture of proteins with molecular masses equal to multiples of 14,300 (lysozyme molecular mass). This mixture can be applied as a set of molecular mass standards suitable for determination of protein molecular masses on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Enhanced detection of glycoproteins in polyacrylamide gels.

A highly sensitive and simple method to enhance detection of glycoproteins resolved by either one- or two-dimensional polyacrylamide gel electrophoresis is described. The method is a modification of the procedure described by D. Fargeaud et al. (D. Fargeaud, J. C. Benoit, F. Kato, and G. Chappuis (1984) Arch. Virol. 80, 69-82) that uses concanavalin A conjugated with fluorescein isothyocyanate to detect the carbohydrate moiety of glycoproteins. Briefly, the electrophoresed gel is exposed to the fluorescent lectin, thoroughly washed, and sequentially transferred to 50% methanol in deionized water and to absolute methanol. The result is an abrupt dehydration of the gel which turns evenly white and stiff. At least a twofold enhancement of fluorescence is obtained as detected by exposing the treated gel to an appropriate uv source. The sensitivity of the procedure allows us to detect purified immunoglobulin molecules by their carbohydrate content in the range of 0.2 microgram of total protein. The specificity of the detection is demonstrated by a comparison with the corresponding polypeptide profile obtained by silver nitrate staining of the gel.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Fluorography of tritium-labeled proteins in silver-stained polyacrylamide gels.

Silver-staining of polyacrylamide gel electrophoresis (PAGE)-separated proteins allows sensitive detection of proteins but severely reduces the ability to detect weak beta-emitters present in the protein band. A simple procedure is described in which silver can be removed from a silver-stained PAGE gel (deargentation) using photographic fixer, and the silver-free gel can be enhanced and used for fluorography. A quantitative study of sensitivity is reported for 3H-labeled bovine serum albumin with a one-dimensional sodium dodecyl sulfate-PAGE slab gel.

Densitometry↗
PubMed1989

Selective silver staining of urease activity in polyacrylamide gels.

A selective method for staining urease activity bands in nondenaturing polyacrylamide gels is described. It is based on the deposition of silver at the urease bands after incubation of gels in the presence of urea and photographic developers. Its highly sensitivity (up to 0.015 enzyme units, corresponding to 5 ng of purified urease) is based on both the silver deposition enhancement methodology and the developers used. The selectivity of the procedure is based on the local pH increase catalytically produced by the enzyme in the presence of urea. The densitometric scan of the enzyme bands gives a linear response at least in the range 0.015-0.300 urease units. This selective staining method is about 2.5 times more sensitive than the standard silver staining of proteins, in terms of detectable urease amount.

Densitometry↗
PubMed1989

Polyacrylamide gel miniaturization improves protein visualization and autoradiographic detection.

Polyacrylamide gels shrink to one-quarter of their original area when soaked in a 50% (w/v) solution of polyethylene glycol. Gel miniaturization improves the contrast of protein bands, with four valuable consequences. (i) A 5- to 10-fold increase in sensitivity for Coomassie blue is observed. (ii) Gels are more durable; i.e., they resist tearing when wet and they do not crack during drying under vacuum. (iii) Shrunken gels give sharper photographic images and provide better interlane protein band comparisons. (iv) Condensed protein bands lead to an increased sensitivity for detecting low-abundance, radioactively-labeled proteins by fluorography.

Affinity Labels↗
PubMed1989

Factors affecting polyacrylamide gel electrophoresis and electroblotting of high-molecular-weight myofibrillar proteins.

Electrophoresis of the high-molecular-mass proteins (greater than 500 kDa) of muscle myofibrils is difficult using conventional procedures. The mobility of these proteins was influenced by the heating time in sample buffer, the use of 2-mercaptoethanol in the upper reservoir buffer, and the pH of the resolving gel in a stacking sodium dodecyl sulfate gel system. Heating samples for 4 min (versus shorter times), addition of 2-mercaptoethanol to the upper reservoir buffer, and reducing the pH of the resolving gel to 8.6 all enhanced the mobility and resolution of the high-molecular-weight proteins on polyacrylamide gels. The sulfhydryl reducing agents commonly used in protein sample buffers (2-mercaptoethanol and dithiothreitol) were found to migrate at the electrophoretic dye front. Inclusion of 10 mM 2-mercaptoethanol in the upper reservoir buffer or blocking free sulfhydryl groups with N-ethylmaleimide prevented intermolecular disulfide bond formation during electrophoresis. The addition of 10 mM 2-mercaptoethanol to the buffer used for electroblotting also improved efficiency of protein transfer to nitrocellulose.

Animals↗
PubMed1989

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.

Electrophoresis, Polyacrylamide Gel↗
PubMed1989

Visualization of acid phosphatase activity on nitrocellulose filters following electroblotting of polyacrylamide gels.

A method for visualizing acid phosphatase isoenzymes by activity staining on nitrocellulose filters after electroblotting of proteins fractionated on nondenaturing polyacrylamide gels is described. Reproducible results were obtained when 25 mM Tris-192 mM glycine was used as the transfer buffer instead of 0.7% acetic acid, 50 mM sodium acetate, pH 4, or 0.14 M acetic acid--0.35 M beta-alanine, pH 4.3. Dot-blot analysis of banana fruit extracts on nitrocellulose filters revealed that a minimum of 5 x 10(-3) units (nmol p-nitrophenyl phosphate hydrolyzed g-1.h-1) of acid phosphatase activity can be detected. This method can be suitable for screening a large number of biological samples for monitoring acid phosphatase activity.

Acid Phosphatase↗
PubMed1989

An optimized procedure for sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of hydrophobic peptides from an integral membrane protein.

A procedure for successful analysis of the hydrophobic tryptic peptides of the Neurospora crassa plasma membrane H+-ATPase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is described. The features of this procedure that are essential for the best results include (i) treatment of the hydrophobic peptide samples with neat trifluoroacetic acid, (ii) dissolution and disaggregation of the hydrophobic peptide samples with SDS at 0 degrees C, (iii) SDS-PAGE of the hydrophobic peptide samples in gels containing a 200:1 ratio of acrylamide to bisacrylamide and a 5-20% convex acrylamide gradient, and (iv) silver-staining of the gels after electrophoresis. This method results in the reproducible resolution and visualization of the H+-ATPase hydrophobic tryptic peptides, which range in size from ca. 5 to 21 kDa, as well as other peptides and proteins ranging in size from ca. 2.5 to 150 kDa. The methods described should also prove useful in other studies where resolution and visualization of hydrophobic peptides of integral membrane proteins are required.

Adenosine Triphosphatases↗
PubMed1989

Two dimensional benzyldimethyl-n-hexadecylammonium chloride----sodium dodecyl sulfate preparative polyacrylamide gel electrophoresis: a high capacity high resolution technique for the purification of proteins from complex mixtures.

A two dimensional preparative polyacrylamide gel electrophoresis system is described which has both high resolving power and high loading capacity (approximately 100 mg protein). The first dimension is electrophoresis toward the cathode at acid pH in the presence of the cationic detergent benzyldimethyl-n-hexadecylammonium chloride (16-BAC), and the second dimension is electrophoresis toward the anode in the presence of sodium dodecyl sulfate. Proteins purified in this way have been sequenced and used successfully as antigens.

Amino Acid Sequence↗
PubMed1989

Enzyme product blot for nondestructive assay of protein catalytic function in polyacrylamide gels.

A new method that permits rapid, sensitive, and specific enzymatic assay of proteins in polyacrylamide gels is described. The enzyme product blot described in this report involves percolation of the reaction mixture through a gel containing native enzyme which converts the labeled substrate to a labeled product with differing chemical properties. A permeable membrane with specific ligand-binding properties overlies the gel and binds the enzyme product, but not the substrate, as reaction mixture is blotted vertically. This membrane is washed free of substrate and the location of the product is identified by autoradiography. The autoradiogram is compared with the stained gel in order to recover the enzyme for amino acid sequence analysis. The enzyme product blot is demonstrated using glycerol kinase and hexokinase.

Autoradiography↗
PubMed1989

Determination of amino acid compositions and NH2-terminal sequences of peptides electroblotted onto PVDF membranes from tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis: application to peptide mapping of human complement component C3.

The combination of high-resolution Tricine-Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (H. Schägger and G. von Jagow (1987) Anal. Biochem. 166, 368-379) and electroblotting onto polyvinylidene difluoride (PVDF) membranes represents a powerful technique for the isolation of small amounts of peptides and protein fragments (Mr 1000-20,000) in a suitable form for amino acid sequencing, directly on the blotting membrane. Conditions for electrophoresis and electroblotting were optimized with respect to high transfer yield and suitability for both amino acid analysis and sequence determination of stained PVDF-bound peptides. Transfer yields were 50-80%, amino acid compositions including Cys were correct, and picomole quantities were sequenced with initial and repetitive yields as high as those we normally obtain for peptides in solution. The method was used for peptide mapping of polymorphic forms of human complement component C3.

Amino Acid Sequence↗
PubMed1989

A method for detection of cellulases in polyacrylamide gels using 5-bromoindoxyl-beta-D-cellobioside: high sensitivity and resolution.

The assay of endo-1,4-beta-glucanases (cellulases) from Trichoderma reesei, T. longibrachiatum, and Sporotrichum pulverulentum by 5-bromoindoxyl-beta-D-cellobioside is described. The substrate is enzymatically cleaved to afford 5-bromoindoxyl and latter undergoes immediate azo coupling with Fast Red or oxidation by nitroblue monotetrazolium chloride, various forms of endoglucanases which can thus be assayed in polyacrylamide gel.

Acrylic Resins↗
PubMed1989

Sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase chromatography of unfolded proteins.

Sequential sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography on a fluorocarbon packing (poly F column, DuPont) provide fully denatured but highly purified protein, which is free of low-molecular-weight substances and directly amenable to structural analysis. Conditions for the preparative elution of four test proteins (bovine serum albumin, carbonic anhydrase, myoglobin, cytochrome c) blotted to Immobilon membranes have been optimized. Phenol saturated with 50 mM Tris-HCl (pH 8.25) and containing 2% SDS and 1% 2-mercaptoethanol is able to elute proteins that have been blotted to Immobilon membranes, stained with Coomassie R-250, and stored as dry sheets, largely irrespective of their molecular mass. Polypeptides that are not degraded by exposure to low pH can also be efficiently extracted directly from stained gels with 70% formic acid. If further separation of polypeptides is not needed, a simple run of a protein sample dissolved in 1-2% SDS on the poly F column will efficiently remove low-molecular-weight substances, including SDS.

Blotting, Western↗
PubMed1989

Solid-phase tyrosine-specific protein kinase assay in multiwell substrate-immobilized polyacrylamide gel.

Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser/Thr-specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compounded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and [tau-32P]ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. 32P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser/Thr-specific protein kinases, can also be analyzed by this technique.

Acrylic Resins↗
PubMed1989

Direct detection of beta-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoelectrofocusing gels.

A procedure to assay isozymes of beta-1,3-glucanase directly on polyacrylamide gel electrophoresis (PAGE) and isoelectrofocusing (IEF) gels by using 2,3,5-triphenyltetrazolium chloride is described. The reagent reacts with reducing sugars released by beta-1,3-glucanases from the substrate laminarin. Acidic and neutral isozymes of beta-1,3-glucanase were detected and quantified on 17.5% native PAGE gels run with an anodic buffer system. A significant linear relationship (alpha = less than 0.01, R = 0.991) was observed between amounts of beta-1,3-glucanase loaded and intensity of bands stained with the reagent on native PAGE gels. A full isozyme pattern was obtained on 7.5% IEF gels with a pH range of 3.5-9.5. The IEF gels were heated in a microwave oven during the staining process to minimize diffusion.

Electrophoresis, Polyacrylamide Gel↗