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

The authors thank the Howard Hughes Medical Institute for a UF Science for Life award to A.H.

1. Preparing the cassettes
<ol>
 <li>Clean front and back plates of a used Invitrogen Nupage Novex minigel cassette, or other commercial minigel cassette.</li>
 <li>Prepare approximately 1-3 ml of plastic epoxy and mix well according to manufacturer's instructions.</li>
 <li>Apply the epoxy to the inside edge of the front plate, where the two plates will come in contact when the cassette is assembled. </li>
 <li>Slide a piece of filter paper or cardboard through the bottom slit of the back plate when assembling the cas...

<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 gel electrophoresis is an indispensible method for most molecular biology laboratories. Commercially available minigels provide a high level of convenience, but can be costly. Here we show how to re-use the minigel cassettes from a popular brand of commercially available minigels. This method retains the convenience of having a ready-made source of gels, but at a fraction of the cost of commercially available minigels. A key step in this procedure is applying the correct amount of epoxy to completely seal the ca...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Invitrogen Nupage Novex Minigel Cassette (1.0 mm)</td>
			<td>Invitrogen</td>
			<td>NC2010</td>
			<td>Available in 2, 5, 10, 12, 15 and 2D wells</td>
		</tr>
		<tr>
			<td>Plastic Epoxy</td>
			<td>Loctite</td>
			<td>108771</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Electrical Tape</td>
			<td>Scotch</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>125 mL side arm flask</td>
			<td>Pyrex</td>
			<td>5360</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>4X Tris-Base resolving gel buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1.5 M Tris-base pH 8.7</td>
		</tr>
		<tr>
			<td>acrylamide </td>
			<td>Fisher Scientific</td>
			<td>01065-500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Bis-acrylamide </td>
			<td>MP Biomedicals, Inc.</td>
			<td>800173</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>30.8% acrylamide solution</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>30:0.8 acrylamide: bis-acrylamide in IRG-H2O</td>
		</tr>
		<tr>
			<td>10% APS</td>
			<td>Fisher Scientific</td>
			<td>BP179-25</td>
			<td>0.1 g ammonium persulfate in 1 ml IRG-H2O, prepared immediately before use</td>
		</tr>
		<tr>
			<td>TEMED</td>
			<td>Arcos Organics</td>
			<td>138450500</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1-butanol saturated with 1X resolving gel buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>5:2 1-butanol to 1X resolving gel buffer</td>
		</tr>
		<tr>
			<td>4X Tris-Base stacking gel buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1.5 M Tris-base pH 6.8</td>
		</tr>
		<tr>
			<td>Gel comb</td>
			<td>Invitrogen</td>
			<td>NC3015</td>
			<td>15 well, 1.0 mm thickness</td>
		</tr>
		<tr>
			<td>Impulse Sealer</td>
			<td>TEW Electric Heating Equipment</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Heat Sealable Pouch</td>
			<td>Kapak/Scotchpak</td>
			<td>&nbsp;</td>
			<td>1 pint size (6.5" x 8")</td>
		</tr>
		<tr>
			<td>Gel storage buffer</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>1X stacking gel buffer with 0.1% sodium azide</td>
		</tr>
		<tr>
			<td>Novex SDS Tris-Glycine sample buffer (2X)</td>
			<td>Invitrogen</td>
			<td>LC2676</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>FisherBiotech</td>
			<td>BP172-5</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>10X Tris-Gylcine Running buffer</td>
			<td>Invitrogen</td>
			<td>LC2675</td>
			<td>1L of 10X stock: 29.0 g Tris-Base, 144.0 g Glycine, 10.0 g SDS</td>
		</tr>
		<tr>
			<td>XCell SureLock Mini-Cell</td>
			<td>Invitrogen</td>
			<td>EI0001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Gel Knife</td>
			<td>Invitrogen</td>
			<td>EI9010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>SimplyBlue Safe Stain</td>
			<td>Invitrogen</td>
			<td>LC6065</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

pouring and running a protein gel by reusing commercial cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

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

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

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

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