Name: pouring and running a protein gel by reusing commercial cassettes
Uploaded: 2012-02-12T14:00:00
Description: Watch this Scientific Journal Video about Pouring and Running a Protein Gel by reusing Commercial Cassettes at JoVE.com

Forfatterne takker Howard Hughes Medical Institute for UF videnskab for Life pris til AH

1. Forberedelse af kassetter <ol><li> Ren og bagpladerne af anvendes Invitrogen Nupage Novex minigel kassette eller andre kommercielle minigel kassette. </li><li> Forberede omkring 1-3 ml af plast epoxy og bland godt i overensstemmelse med producentens instruktioner. </li><li> Anvende epoxy til den indvendige kant af den forreste plade, hvor de to plader vil komme i kontakt, når kassetten er samlet. </li><li> Glide et stykke filtrerpapir eller karton gennem bunden slids bagpladen ved montering af kassetten for at for...

<ol>
	<li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227, 680-685 (1970).</li><li>Raymond, S., Weintraub, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acrylamide+gel+as+a+supporting+medium+for+zone+electrophoresis.">Acrylamide gel as a supporting medium for zone electrophoresis.</a> Science. 130, 711-711 (1959).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Shapiro, A. L., Vi&#241;uela, E., Maizel, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+weight+estimation+of+polypeptide+chains+by+electrophoresis+in+SDS-polyacrylamide+gels.">Molecular weight estimation of polypeptide chains by electrophoresis in SDS-polyacrylamide gels.</a> Biochem. Biophys. Res. Commun. 28, 815-820 (1967).</li></ol>

Protein gelelektroforese er en uundværlig fremgangsmåde til fleste molekylærbiologiske laboratorier. Kommercielt tilgængelige minigelerne sikre et højt niveau af bekvemmelighed, men kan være dyrt. Her viser vi, hvordan man genbruge de minigel kassetter fra et populært mærke af kommercielt tilgængelige minigelerne. Denne fremgangsmåde bevarer bekvemmelighed har en færdiglavet kilde geler, men en brøkdel af omkostningerne ved kommercielt tilgængelige minigeler. Et vigtigt trin i denne procedure anvender den k...

<table border="1"><tbody><tr><td> Navn reagens </td><td> Firma </td><td> Katalog nummer </td><td> Kommentarer </td></tr><tr><td> Invitrogen Nupage Novex minigel kassette (1,0 mm) </td><td> Invitrogen </td><td> NC2010 </td><td> Fås i 2, 5, 10, 12, 15 og 2D brønde </td></tr><tr><td> Plastic Epoxy </td><td> Loctite </td><td> 108771 </td><td></td></tr><tr><td> Elektrisk Tape </td><td> Skotsk whisky </td><td></td><td></td></tr><tr><td> 125 ml sidearm kolbe </td><td> Pyrex </td><td> 5360 </td><td></td></tr><tr><td> 4X Tris-base opløsende gel buffer </td><td></td><td></td><td> 1,5 M Tris-base pH 8,7 </td></tr><tr><td> acrylamid </td><td> Fisher Scientific </td><td> 01065-500 </td><td></td></tr><tr><td> Bis-acrylamid </td><td> MP Biomedicals, Inc. </td><td> 800173 </td><td></td></tr><tr><td> 30,8% acrylamid opløsning </td><td></td><td></td><td> 30:0.8 acrylamid: bis-acrylamid i IRG-H2O </td></tr><tr><td> 10% APS </td><td> Fisher Scientific </td><td> BP179-25 </td><td> 0,1 g ammoniumpersulfat i 1 ml IRG-H2O, fremstillet umiddelbart før brug </td></tr><tr><td> TEMED </td><td> Arcos Organics </td><td> 138450500 </td><td></td></tr><tr><td> 1-butanol mættet med 1X opløsende gel buffer </td><td></td><td></td><td> 05:02 1-butanol til 1X opløsende gel buffer </td></tr><tr><td> 4X Tris-basestabling gelpuffer </td><td></td><td></td><td> 1,5 M Tris-base pH 6,8 </td></tr><tr><td> Gel kam </td><td> Invitrogen </td><td> NC3015 </td><td> 15 brønd, 1,0 mm tykkelse </td></tr><tr><td> Impulsforseglingsorgan </td><td> TEW Elektrisk Varme Udstyr </td><td></td><td></td></tr><tr><td> Varmeforseglelig pose </td><td> Kapak / Scotchpak </td><td></td><td> 1 pint størrelse (6,5 &quot;x 8&quot;) </td></tr><tr><td> Gel opbevaringspuffer </td><td></td><td></td><td> 1X stacking gel buffer med 0,1% natriumazid </td></tr><tr><td> Novex SDS Tris-Glycin-prøvebuffer (2X) </td><td> Invitrogen </td><td> LC2676 </td><td></td></tr><tr><td> DTT </td><td> FisherBiotech </td><td> BP172-5 </td><td></td></tr><tr><td> 10X Tris-Gylcine løbende buffer </td><td> Invitrogen </td><td> LC2675 </td><td> 1L af 10X rådighed: 29,0 g Tris-base, 144,0 g glycin, 10,0 g SDS </td></tr><tr><td> XCell SureLock Mini-Cell </td><td> Invitrogen </td><td> EI0001 </td><td></td></tr><tr><td> Gel Knife </td><td> Invitrogen </td><td> EI9010 </td><td></td></tr><tr><td> SimplyBlue Sikker Stain </td><td> Invitrogen </td><td> LC6065 </td&gt; <td></td></tr></tbody></table>

pouring and running a protein gel by reusing commercial cassettes

Evaluering af proteiner under anvendelse af natriumdodecylsulfat-polyacrylamidgelelektroforese (SDS-PAGE-analyse) er en almindelig teknik ved biokemiske og molekylærbiologiske forskere 1-4. For laboratorier, som udfører daglige analyser af proteiner, kan omkostningerne ved kommercielt tilgængelige polyacrylamidgeler (~ $ 10/gel) være betydeligt med tiden. For at mindske denne omkostning, nogle forskere forberede deres egne polyacrylamidgeler. Traditionelle metoder til at hælde disse geler typisk anvender specialudstyr og glas gel plader, der kan være dyrt og udelukker hælde mange geler og gemme dem til senere brug. Derudover kan håndtering af glasplader under rengøring eller gel hælde resultere i utilsigtet brud skabe en sikkerhedsrisiko, som kan udelukke deres anvendelse i bachelor laboratorie klasser. Vores protokol viser, hvordan man hælde flere proteingeler samtidig ved genbrug Invitrogen Nupage Novex minigel kassetter, og billig marialer købt på et hjem forbedring butik. Dette økonomiske og strømlinet Fremgangsmåden indbefatter en måde at opbevare gelerne ved 4 ° C i nogle få uger. Ved genbrug af plastic gelkassetter fra kommercielt tilgængelige geler, laboratorier, der kører hyppige proteingeler kan spare betydelige omkostninger og hjælpe miljøet. Desuden er plast gelkassetterne ekstremt modstandsdygtige over for beskadigelse, hvilket gør dem ideelle til undergraduate laboratorie klasseværelser.

Watch this Scientific Journal Video about Pouring and Running a Protein Gel by reusing Commercial Cassettes at JoVE.com

Pouring and Running a Protein Gel by reusing Commercial Cassettes

Vores protokol viser, hvordan man hælde flere proteingeler ad gangen ved genbrug Invitrogen Nupage Novex minigel kassetter og billige materialer, der indkøbes på et hjem forbedring butik. Dette økonomiske og strømlinet Fremgangsmåden indbefatter en måde at opbevare gelerne ved 4 ° C i nogle få uger. Ved genbrug af plastic gelkassetter fra kommercielt tilgængelige geler, laboratorier, der kører hyppige proteingeler kan spare betydelige omkostninger og hjælpe miljøet.

Hælde og køre en proteingel ved at genbruge Commercial Kassetter

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Research

JoVE Journal

Biology

Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis DGGE

Screening for Amyloid Aggregation by Semi-Denaturing Detergent-Agarose Gel Electrophoresis

Amyloid aggregation is associated with numerous protein misfolding pathologies and underlies the infectious properties of prions, which are ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Ortogonale Proteinoprensning Faciliteret af en lille Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Forbedring succesraten for proteinkrystallisation af Random Microseed Matrix Screening

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Protein-protein interaktioner visualiseret ved bimolekylær Fluorescens komplementering i tobaksprotoplaster og blade

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional sporing af hæmatopoietisk stamcelletransplantation og stamcelle i Voksen Mouse calvarie knoglemarv

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

Grønt fluorescerende protein-baserede Expression Screening membranproteiner i<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...