Rapid visuallzation of protein-dodecyl sulfate complexes in polyacrylamide gels

Nelles, Lynn P.; Bamburg, James R.

doi:10.1016/0003-2697(76)90202-5

Cited by 97 publications

(31 citation statements)

References 16 publications

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“…rcClCBase purified from inclusion bodies by using an immobilized metal affinity column under denaturing conditions was further purified to homogeneity by using 10% SDS-PAGE, nonfixing KCl staining (36), and gel elution at 5 mA for 16 h using an electroeluter (Bio-Rad) as described in the instruction manual. The purified rcClCBase, which was more than 98% pure as determined by SDS-PAGE with colloidal Coomassie blue staining, was used for production of antibodies.…”

Section: Methodsmentioning

confidence: 99%

Outer Membrane Proteins ofFibrobacter succinogeneswith Potential Roles in Adhesion to Cellulose and in Cellulose Digestion

et al. 2007

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Comparative analysis of binding of intact glucose-grown Fibrobacter succinogenes strain S85 cells and adhesion-defective mutants AD1 and AD4 to crystalline and acid-swollen (amorphous) cellulose showed that strain S85 bound efficiently to both forms of cellulose while mutant Ad1 bound to acid-swollen cellulose, but not to crystalline cellulose, and mutant Ad4 did not bind to either. One-and two-dimensional electrophoresis (2-DE) of outer membrane cellulose binding proteins and of outer membranes, respectively, of strain S85 and adhesion-defective mutant strains in conjunction with mass spectrometry analysis of tryptic peptides was used to identify proteins with roles in adhesion to and digestion of cellulose. Examination of the binding to cellulose of detergent-solubilized outer membrane proteins from S85 and mutant strains revealed six proteins in S85 that bound to crystalline cellulose that were absent from the mutants and five proteins in Ad1 that bound to acid-swollen cellulose that were absent from Ad4. Twenty-five proteins from the outer membrane fraction of cellulose-grown F. succinogenes were identified by 2-DE, and 16 of these were up-regulated by growth on cellulose compared to results with growth on glucose. A protein identified as a Cl-stimulated cellobiosidase was repressed in S85 cells growing on glucose and further repressed in the mutants, while a cellulose-binding protein identified as pilin was unchanged in S85 grown on glucose but was not produced by the mutants. The candidate differential cellulose binding proteins of S85 and the mutants and the proteins induced by growth of S85 on cellulose provide the basis for dissecting essential components of the cellulase system of F. succinogenes.Fibrobacter succinogenes S85 is a rod-shaped, gram-negative, strictly anaerobic ruminal bacterium that is one of the major cellulolytic bacteria within the rumen (2, 24). Because of the ability of this bacterium to efficiently adhere to and degrade plant cell walls, it has been extensively studied to elucidate the mechanism involved in adhesion and digestion of plant cell walls. As a result, five endoglucanases (31, 33-35), one cellobiosidase (17), one cellodextrinase (15), four xylanases (21, 52), two acetyl xylan esterases (22), and two cellulose-binding proteins (11) have been purified or cloned and purified and the catalytic properties of the enzymes determined. Although those studies have provided valuable information about many of the cellulases and hemicellulases of F. succinogenes, the mechanism of binding of cells to cellulose and hydrolysis of cellulose has not been solved.Similar to the case with other anaerobic cellulolytic bacteria, adhesion of F. succinogenes cells to cellulose appears to be a prerequisite for rapid and efficient cellulose hydrolysis by this organism (30). This claim is supported by the observation that carboxymethylcellulose, which blocks adhesion to cellulose, also blocked cellulose degradation (25). Gong and Forsberg (12) isolated nonadherent mutants of F. succinogenes S85 tha...

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Section: Methodsmentioning

confidence: 99%

Outer Membrane Proteins ofFibrobacter succinogeneswith Potential Roles in Adhesion to Cellulose and in Cellulose Digestion

et al. 2007

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“…The SDS-containing discontinuous buffer system by Neville (1971) (Nelles & Bamburg, 1976), cut out of the gel, dialysed against distilled water for 24h and homogenized. The homogenate was emulsified with Freund's complete adjuvant and injected into one lymph node on the back side of each thigh of a rabbit.…”

Section: Methodsmentioning

confidence: 99%

Properties of salt-resistant lipase and lipoprotein lipase purified from human post-heparin plasma

Ostlund-Lindqvist¹

1979

Biochemical Journal

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Lipoprotein lipase and salt-resistant lipase were isolated from human post-heparin plasma. The proteins of human post-heparin-plasma lipoprotein lipase and salt-resistant lipase were identified and demonstrated to be immunologically different. Significant differences between the two enzymes in their relative amino acid composition were demonstrated, which indicates that the two enzymes are different proteins. When analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the enzymes seemed to have monomer molecular weights similar to that of lipoprotein lipase purified from bovine milk.

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“…Material in the electrophoresis gel is detectable to less than 100 pmol by a modified KCI visualization [12], corresponding to all bands that would be clearly visible by ordinary Coomassie brilliant blue staining. Results are interpretable in all cases studied where the polypeptide investigated has a free N-terminus.…”

Section: Discussionmentioning

confidence: 99%

“…Gentle rocking showed protein bands within approximately 2 min, as clear zones against an opaque background (modified from [12] by omission of acetic acid since fixation is not desirable). Detection is facilitated by visualization in a dark room with a light source from obliquely below.…”

Section: Detection and Excision Of Discrete Protein Bandsmentioning

confidence: 99%

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

Bergman

Jörnvall

1987

Eur J Biochem

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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 (1 2 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.Blotting of proteins onto glass-fiber filters, in a manner related to the widely used Western blotting onto nitrocellulose membranes [I], is valuable for direct analysis by gas-phase sequencer degradations after electrophoretic separation on SDS/polyacrylamide gels [2]. Variations in size of filters, type of pretreatment, and details of the blotting have been described [2, 31, but in common these methods have transfer of all proteins occurring on a gel, followed by localizations on the filter by Coomassie or fluorescence staining techniques. We have tested a method avoiding staining and allowing transfer of individual bands onto filters of a size suitable for direct use in a sequencer (Fig.

show abstract

Rapid visuallzation of protein-dodecyl sulfate complexes in polyacrylamide gels

Cited by 97 publications

References 16 publications

Outer Membrane Proteins ofFibrobacter succinogeneswith Potential Roles in Adhesion to Cellulose and in Cellulose Digestion

Outer Membrane Proteins ofFibrobacter succinogeneswith Potential Roles in Adhesion to Cellulose and in Cellulose Digestion

Properties of salt-resistant lipase and lipoprotein lipase purified from human post-heparin plasma

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

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