scholarly journals Quantificação de [32P] fosfoproteínas em gel de pollacrilamida pela Radiação Cerenkov: vantagens sobre outros procedimentos

1997 ◽  
Vol 19 (19) ◽  
pp. 21
Author(s):  
Carlos A. Gonçalves ◽  
Guido Lenz ◽  
Christianne Salbego ◽  
Carmem Gottfried
Keyword(s):  
Liquid Scintillation Counting ◽  
Gel Electrophoresis ◽  
Liquid Scintillation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Scintillation Counting ◽  
Polyacrylamide Gel ◽  
Cerenkov Counting ◽  
Phosphorylation Of Proteins ◽  
Phosphate Incorporation

The phosphorylation of proteins has been recognized as an important mechanism for controlling cellular activities. There are many ways to measure 32P-labellingof phosphoproteins resolved by polyacrylamide gel electrophoresis, including densitometry of autoradiographs, liquid scintillation counting and Cerenkov counting. This report compares such different procedures and indicates the advantages of Cerenkov counting to determine radioactive phosphate incorporation into proteins.

Purification of Labeled Oligonucleotides by Size-Exclusion Chromatography

2021 ◽  
Vol 2021 (10) ◽  
pp. pdb.prot100719
Author(s):  
Michael R. Green ◽  
Joseph Sambrook
Keyword(s):  
Size Exclusion Chromatography ◽  
Liquid Scintillation Counting ◽  
Gel Electrophoresis ◽  
Liquid Scintillation ◽  
Scintillation Counting ◽  
Size Exclusion ◽  
Enzymatic Reactions ◽  
Cetylpyridinium Bromide ◽  
Exclusion Chromatography ◽  
Differential Precipitation

When labeled oligonucleotides are to be used in enzymatic reactions such as primer extension, virtually all of the unincorporated label must be removed from the oligonucleotide. For this purpose, chromatographic methods or gel electrophoresis are superior to differential precipitation of the oligonucleotide with ethanol or cetylpyridinium bromide (CPB). This protocol describes a method to separate labeled oligonucleotides from unincorporated label that takes advantage of differences in mobility between oligonucleotides and mononucleotides during size-exclusion chromatography. Although size-exclusion chromatography can, in principle, be used to purify either radiolabeled or nonradiolabeled oligonucleotides, this protocol is geared toward purifying radiolabeled oligonucleotides, whose elution from the column is monitored using a minimonitor and whose separation from unincorporated nucleotides is monitored by liquid scintillation counting.

scholarly journals Non-Specific Binding of Thiocholine Ester of Cinnamic Acid to Fibrin

1977 ◽  
Author(s):  
T. Seelich ◽  
B. A. Perret ◽  
M. Furlan ◽  
E. A. Beck
Keyword(s):  
Cinnamic Acid ◽  
Gel Electrophoresis ◽  
Liquid Scintillation ◽  
Specific Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Scintillation Counting ◽  
Human Fibrinogen ◽  
Α Chain ◽  
Β Chain

Thiocholine esters inhibit the enzymatic crosslinking of fibrin (1). Thiocholine ester of 14C-labelled cinnamic acid (2-diethylbenzylaminoethyl thioltranscinnamate bromide) was prepared and incubated with human fibrinogen in the presence of factor XIII and thrombin. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate, followed by liquid scintillation counting of the separated protein bands, showed that all three chains of fibrinogen had bound the crosslinking inhibitor(2 moles/mol α chain, 1.5 moles/mol β chain, 2.2 moles/mol γ chain). Cyanogen bromide cleavage of the isolated α chain resulted in 6 major fragments all of whichwere radioactive. These results indicate that the binding of the thiocholine ester of cinnamic acid was not restricted to the specific donor crosslinking sites of fibrin(ogen). This thiocholine ester differs in this respect from dansyl-cadaverine which binds exclusively to the acceptor sites involved in fibrin(ogen) crosslinking.

scholarly journals Turnover of acyl-CoA-binding protein in four different cell lines measured by using two-dimensional polyacrylamide-gel electrophoresis

1994 ◽  
Vol 297 (3) ◽  
pp. 555-560 ◽  
Author(s):  
C L Buus ◽  
K Kristiansen ◽  
J Knudsen
Keyword(s):  
Cell Lines ◽  
Total Protein ◽  
Gel Electrophoresis ◽  
Liquid Scintillation ◽  
Binding Protein ◽  
Biological Activities ◽  
Polyacrylamide Gel Electrophoresis ◽  
Growth Conditions ◽  
Scintillation Counting ◽  
Two Dimensional

Acyl-CoA-binding protein (ACBP), also named diazepam-binding inhibitor or endozepine, is a 10 kDa protein for which a surprisingly large number of biological activities has been suggested. Some of these would seem to require a rapid intracellular turnover of the protein. In this paper we report on the turnover of ACBP in cell lines derived from mouse, rat and man. ACBP was identified in two-dimensional gels by using specific antibodies. Cells were labelled with [35S]methionine and chased for various periods of time. Total protein was extracted, subjected to two-dimensional PAGE, and radioactivity in the spot containing ACBP was determined by liquid-scintillation counting. ACBP half-life was determined, and varied from 25 to 53 h depending on the cell line and the growth conditions. In all cases, radioactivity in ACBP was lost slightly faster than radioactivity in total protein. These results are discussed in relation to the possible function suggested for ACBP.

Ultrastructural localization of specific nucleoprotein fractions

1978 ◽  
Vol 36 (2) ◽  
pp. 204-205
Author(s):  
G. L. Brown
Keyword(s):  
Gel Electrophoresis ◽  
Mouse Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Ultrastructural Localization ◽  
Polyacrylamide Gel ◽  
Acid Extraction ◽  
Acid Solutions ◽  
Low Salt ◽  
Liver Nuclei ◽  
Phosphate Groups

Bismuth (Bi) stains nucleoproteins (NPs) by interacting with available amino and primary phosphate groups. These two staining mechanisms are distinguishable by glutaraldehyde crosslinking (Fig. 1,2).Isolated mouse liver nuclei, extracted with salt and acid solutions, fixed in either formaldehyde (form.) or gl utaraldehyde (glut.) and stained with Bi, were viewed to determine the effect of the extractions on Bi stainina. Solubilized NPs were analyzed by SDS-polyacrylamide gel electrophoresis.Extraction with 0.14 M salt does not change the Bi staining characteristics (Fig. 3). 0.34 M salt reduces nucleolar (Nu) staining but has no effect on interchromatinic (IC) staining (Fig. 4). Proteins responsible for Nu and glut.- insensitive IC staining are removed when nuclei are extracted with 0.6 M salt (Fig. 5, 6). Low salt and acid extraction prevents Bi-Nu staining but has no effect on IC staining (Fig. 7). When nuclei are extracted with 0.6 M salt followed by low salt and acid, all Bi-staining components are removed (Fig. 8).

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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