A Fast and Inexpensive Western Blot Experiment for the Undergraduate Laboratory

This paper describes a mechanism of cell proliferation promotion of cultured osteoblasts by mechanical vibration focusing on β-catenin. 12.5 Hz and 0.5 G mechanical vibration was reported to promote the cell proliferation of cultured osteoblasts in plane culture. That is because the mechanical vibration weakens cell-cell adhesion, promotes to pile up cells, and allows cells to form multilayer structure. However, it has not been clarified why cells continue cell division after their monolayer confluent state. Here we show that mechanical vibration not only weakens cell-cell adhesion bound by β-catenin but also promotes to move β-catenin from the cytoplasm to the nuclei, where β-catenin associates with DNA-binding members of the Tcf/LEF family and other associated transcription factors including cell division. After osteoblastic cells were cultured under 12.5 Hz and 0.5 G mechanical vibration, cells were fractionated into nuclear and cytoplasmic fractions using a centrifugation method. β-catenin in each fraction was detected by a western blot experiment. The protein bands from western blot films were quantified with an image processing and analysis software, ImageJ. As a result, the vibration group gave higher expression of β-catenin in nuclear fraction than the non-vibration group just after the vibration group reached the saturated cell density. It indicates that 12.5 Hz and 0.5 G mechanical vibration may promote to move β-catenin into the nuclei and the cell division.

The Design of a Quantitative Western Blot Experiment

BioMed Research International ◽

10.1155/2014/361590 ◽

2014 ◽

Vol 2014 ◽

pp. 1-8 ◽

Cited By ~ 108

Author(s):

Sean C. Taylor ◽

Anton Posch

Keyword(s):

Western Blot ◽

Western Blotting ◽

Dynamic Range ◽

Complex Mixture ◽

Experimental Methodology ◽

Western Blots ◽

Western Blot Experiment ◽

Fundamental Shift ◽

Western Blot Data ◽

Densitometric Data

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting.

A Convenient and Highly Specific Western Blot Experiment for Introductory Biochemistry

Journal of Chemical Education ◽

10.1021/ed077p373 ◽

2000 ◽

Vol 77 (3) ◽

pp. 373 ◽

Cited By ~ 4

Author(s):

Donald G. Gerbig ◽

Christopher J. Fenk ◽

Stephanie Y. Grooms

Keyword(s):

Western Blot ◽

Western Blot Experiment

Vorkommen von Antikörpern gegen das bovine Immunschwächevirus bei Rindern in Bayern

Tierärztliche Praxis Ausgabe G Großtiere / Nutztiere ◽

10.1055/s-0038-1622979 ◽

2003 ◽

Vol 31 (05) ◽

pp. 248-253

Author(s):

Maren Bartels ◽

Katrin Hartmann ◽

L. Scobie ◽

O. Jarrett ◽

W. Klee

Keyword(s):

Western Blot

ZusammenfassungIm Rahmen einer epidemiologischen Untersuchung zur Infektion mit dem bovinen Immunschwächevirus (BIV) bei Rindern in Oberbayern erfolgten zwei Studien, in denen Serum mittels indirektem ELISA auf BIV-Antikörper untersucht wurde. Die ELISA-Ergebnisse der BIV-positiven Tiere der Studie I wurden mittels Western Blot bestätigt. In Studie I wurde Blut von 173 ungezielt ausgewählten Rinderpatienten der II. Medizinischen Tierklinik der Ludwig-Maximilians-Universität München untersucht. Von diesen waren acht Tiere BIV-infiziert. Das entspricht einer Prävalenz von 4,6%. Alle positiven Tiere waren über zwei Monate alt. In Studie II wurden 550 Kühe aus 11 oberbayerischen landwirtschaftlichen Betrieben untersucht. Hiervon waren 11 Tiere BIVAntikör-perpositiv. Dies entspricht einer Prävalenz von 2,0%. Die positiven Tiere stammten aus fünf Betrieben mit Boxenlaufstallhaltung. Kein Tier aus Betrieben mit Anbindehaltung war positiv. In Studie II lag das Durchschnittsalter der Kühe aus den Betrieben ohne BIV-infizierte Tiere signifikant höher als in den Betrieben mit BIV-infizierten Tieren. Die Prävalenz von BIV-Antikörpern war zwar unter den kranken Probanden aus Studie I signifikant höher als bei den klinisch unauffälligen Rindern der Studie II, die pathogene Bedeutung des BIV erscheint jedoch fraglich.

Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

Western Blot Analyses of Prekallikrein and its Activation Products in Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648157 ◽

1991 ◽

Vol 65 (04) ◽

pp. 382-388 ◽

Cited By ~ 9

Author(s):

Dulce Veloso ◽

Robert W Colman

Keyword(s):

Complex Formation ◽

Western Blot ◽

Human Plasma ◽

Contact System ◽

Plasma Activation ◽

Normal Plasma ◽

Sds Page ◽

Activation Products ◽

Major Antigen ◽

Formation Of Complexes

SummaryPrekallikrein (PK), a zymogen of the contact system, and its activation products, kallikrein (KAL), KAl-inhibitor complexes and fragments containing KAL epitope(s) have been detected in human plasma by immunoblotting with a monoclonal anti-human plasma PK antibody, MAb 13G1L. Detection of antigen-MAb 13G11 complexes with peroxidase-conjugated anti-IgG showed that the two variants of PK (85- and 88-kDa) are the only major antigen species in normal, non-activated plasma. Upon plasma activation with kaolin, the intensity of the PK bands decreased with formation of complexes of KAL with CL inhibitor (C1 INH) and α2-rrtzcroglobulin (α2M) identical to those formed by the purified proteins. Immunoblots of normal plasma showed good correlation between the PK detected and the amount of plasma assayed. Increasing amounts of KAL incubated with a constant volume of PK-deficient plasma showed increasing amounts of KAL and of KAL-C1 INH and KAL-α2M complexes. Complexes of KALantithrombin III (ATIII) and the ratio of KALα2M/ KAL-CL INH were higher in activated CL INH-deficient plasmas than in activated normal plasmas. Protein resolution by 3-12% gradient SDS-PAGE and epitope detection with [125I]MAb 13G11 showed four KALα2M species and a 45-kDa fragment(s) in both surface-activated normal plasma and complexes formed by purified KAL and α2M. Immunoblots of activated plasma also showed bands at the position of KALCL INH and KALATIII complexes. When α1-antitrypsin Pittsburgh (cα1-AT, Pitts) was added to plasma before activation, KAL-α1-ALPitts was the main complex. The non-activated normal plasma revealed only an overloaded PK band. This is the first report of an antibody that recognizes KAL epitope(s) in KAL-α2M, KALATIII and KALa1-α1Pitts complexes and in the 45-kDa fragment(s). Therefore, MAb 13G11 should be useful for studying the structure of these complexes as well as the mechanism of complex formation. In addition, immunoblotting with MAb 13G11 would allow detection of KAl-inhibitor complexes in patient plasmas as indicators of activation of the contact system.

Differences in the Interactions of Lupus Anticoagulant IgG with Human Prothrombin and Bovine Prothrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1653838 ◽

1995 ◽

Vol 73 (04) ◽

pp. 668-674 ◽

Cited By ~ 34

Author(s):

L Vijaya Mohan Rao ◽

An D Hoang ◽

Samuel I Rapaport

Keyword(s):

Western Blot ◽

Lupus Anticoagulant ◽

Protein A ◽

Phospholipid Complex ◽

Experimental Conditions ◽

Bovine Plasma ◽

Activation System ◽

Rate Limiting ◽

Substantial Extent ◽

Prothrombin Activation

SummaryLupus anticoagulant (LA) IgGs have been reported to inhibit more effectively and consistently the Xa/Va/phospholipid complex-catalyzed activation of human prothrombin than the Xa/Va/phospholipid complex-catalyzed activation of bovine prothrombin. This led us to carry out studies to determine whether the ability to inhibit the activation of prothrombin of LA IgGs, separated from the plasma of 15 patients by protein A affinity chromatography, could be related to the ability of the LA IgGs to bind to prothrombin under various experimental conditions. Of 14 LA IgG preparations tested all prolonged to a variable but substantial extent the dilute Russell’s viper venom time (dRVVT) of human plasma but only minimally prolonged the dRVVT of bovine plasma. In a purified prothrombin activation system with a rate limiting concentration of phospholipid, all 15 LA IgG preparations inhibited the activation of human prothrombin with the majority showing >50% of inhibition. In contrast, only one LA IgG markedly inhibited (>50%) the activation of bovine prothrombin and five others moderately inhibited (25-40%) the activation of bovine prothrombin. Nevertheless, the majority of LA IgG preparations bound to immobilized bovine prothrombin on a Western blot and also to immobilized bovine prothrombin on a microtiter well. In an ELISA in which phosphatidylserine (PS) was immobilized on microtiter wells, bovine prothrombin supported the binding of 10 of 15 LA IgG preparations to PS. However, the extent of binding was lower than that observed with human prothrombin. In experiments with 125I-human prothrombin or 125I-bovine prothrombin in a solution containing Ca2+, the addition of PS/PC vesicles enhanced the binding of both human and bovine prothrombin to some LA IgG preparations. The enhanced binding was particularly evident for bovine prothrombin. Although seemingly related for some preparations, the ability of a LA IgG to bind to bovine prothrombin, either in the presence or absence of PS, and the ability of that LA IgG to inhibit the activation of bovine prothrombin was not consistently related for all preparations.

The Development of An Undergraduate Laboratory for Control System Design

1993 American Control Conference ◽

10.23919/acc.1993.4793234 ◽

1993 ◽

Cited By ~ 1

Author(s):

Garng M. Huang ◽

Li-Fang Fu ◽

John Fleming

Keyword(s):

Control System ◽

System Design ◽

Control System Design ◽

Undergraduate Laboratory

Early postnatal diagnosis of congenital syphilis: contribution of a comparative Western Blot method

10.26226/morressier.56d5ba31d462b80296c967a7 ◽

2016 ◽

Author(s):

Antonella Marangoni

Keyword(s):

Western Blot ◽

Congenital Syphilis ◽

Western Blot Method ◽

Postnatal Diagnosis