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 cassette to prevent excess epoxy from sealing the opening. Remove the filter paper as soon as the two plates are assembled.</li>
 <li>Place binder clips along the edges for a tight seal, and let the cassettes sit overnight for the epoxy to fully cure.</li>
 <li>Seal the slit on the bottom of the back plate with electrical tape. </li>
</ol>
2. Pouring the resolving gel
<ol>
 <li>To a 125 ml side arm flask, add the following: 2.25 ml of 4X Tris-Base Resolving gel buffer pH 8.70, 3.50 ml of 30.8% acrylamide/bisacrylamide, 3.25 ml of Type I reagent grade water (IRG-H2O) for a total volume of 9 ml.</li>
 <li>De-gas the solution for 10 min by attaching the side arm of the flask to a vacuum apparatus, and placing an inverted stopper on the top of the flask.</li>
 <li>Transfer the 9 ml of de-gassed solution to a 50 ml disposable polypropylene centrifuge tube and add 30 &mu;l of 10% ammounium persulfate (APS) and 6 &mu;l of N, N, N', N',-tetramethylethylenediamine (TEMED) and mix well by swirling the solution to avoid introducing bubbles. </li>
 <li>Immediately pour the solution into the cassette, allowing it to run down the side of the cassette to prevent any bubbles from forming between the gel plates. Fill the cassette to approximately 2 cm from the top edge of the front (shorter) plate. </li>
 <li>Gently add 0.5 ml of 1-butanol saturated with 1X resolving gel buffer and let the gel polymerize for 1 hour.</li>
</ol>
3. Pouring the stacking gel
<ol>
 <li>Add the following to a 125 ml side arm flask: 0.75 ml of 4X Tris-Base Stacking gel buffer pH 6.80, 0.40 ml of 30.8% acrylamide/bisacrylamide, 1.85 ml of IRG-H2O for a total volume of 3 ml.</li>
 <li>De-gas the solution for 10 min as described in step 2.</li>
 <li>While the stacking gel solution is degassing, pour off the 1-butanol saturated with 1X resolving gel buffer from the top of the resolving gel. Rinse the top of the resolving gel once with IRG-H2O. Pour off the water and use a bibulous or Whatman filter paper to absorb any remaining liquid.</li>
 <li>Remove 3 ml of your degassed solution and add 9 &mu;l of 10% APS and 2 &mu;l of TEMED and mix well.</li>
 <li>Quickly pour the solution in the cassette, allowing it to run down the side of the cassette until it is near the top edge of the shorter front gel plate.</li>
 <li>Insert a clean gel comb into the top of the gel cassette, place a binder clip on the cassette over the comb to hold it in place. Let the stacking gel polymerize for 1 hour to overnight.</li>
</ol>
4. Storing the gel
<ol>
 <li>When the gel has completely polymerized, remove the binder clips and place the cassette in a heat-sealable plastic pouch, or a re-sealable plastic bag.</li>
 <li>Pour 20 ml of 1X stacking gel buffer that contains 0.1% sodium azide into the pouch.</li>
 <li>Heat seal the pouch for 5 sec and make sure there are no leaks in the bag.</li>
 <li>Store the gel at 4&deg;C.</li>
</ol>
5. Preparing the Samples
<ol>
 <li>In a thin-walled microcentrifuge tube, add up to 6.5 &mu;l of protein sample, 7.5 &mu;l of 2x Novex Tris-Glycine SDS sample buffer, 1 &mu;l of 100 mM dithiothreitol (DTT) and IRG-H2O (if necessary) for a total volume of 15 &mu;l.</li>
<li>Mix the sample by vortexing, and briefly centrifuge the sample to bring contents to the bottom of the tube.</li>
 <li>Denature the sample at 70&deg;C for 10 min.</li>
</ol>
6. Running the gel
<ol>
 <li>Set up the Novex gel apparatus. </li>
 <li>Add 200 ml of Tris-Glycine Running buffer that contains 500 &mu;l of Nupage antioxidant to the inner chamber, and 300 ml of Tris-Glycine Running buffer to the outer chamber.</li>
 <li>Load the denatured protein samples (15 &mu;l) into the wells using a gel loading pipette tip.</li>
 <li>Run the gel at 200 V for 60-70 min.</li>
</ol>
7. Staining the gel
<ol>
 <li>Remove the cassette from the gel apparatus and break the epoxy seal by prying the two plastic gel plates apart using a gel knife or a stiff putty knife. Carefully separate the gel from the plates, taking care to not tear the gel.</li>
 <li>Rinse the gel with IRG-H2O three times with gentle agitation.</li>
 <li>Stain the gel with Invitrogen SimplyBlue SafeStain for 1 hour with gentle agitation.</li>
 <li>Pour off the Staining solution and destain the gel with IRG-H2O.</li>
</ol>
8. Cleaning the cassettes
<ol>
 <li>Use a metal spatula to scrape or peel off all the epoxy on the cassettes so they can be reused again. </li>
 <li>Wash each of the plastic gel plates with a mild detergent solution, and rinse well with tap water followed by distilled water.</li>
</ol>
9. Representative Results
Because epoxy will form a water-tight seal, (Figure 1), the cassettes will not leak when the acrylamide solution is poured into them. In addition, the gel cassettes can be easily opened to retrieve the gel for staining, and the epoxy can be peeled off of the cassettes to allow their continued re-use. As shown in Figure 2, the gel shows clear separation of protein markers and sample bands suitable for routine protein electrophoresis.
<img src="/files/ftp_upload/3465/3465fig1.jpg" alt="Figure 1" /> 
Figure 1. Application of epoxy to seal minigel cassettes. A thin layer of epoxy is applied to the edge of one of the cassettes prior to assembly. To highlight the epoxy, it was false-colored red in this image. 
<img src="/files/ftp_upload/3465/3465fig2.jpg" alt="Figure 2" /> 
Figure 2. Separation of proteins on an SDS-Tris-glycine gel poured using re-used minigel cassettes. SDS-PAGE was performed as described above. Following electrophoresis the gel was removed from the minigel cassette, stained in SimplyBlue SafeStain as per manufacturer's instructions, and photographed using transmitted white light. Lane M contains SeeBlue Plus2 pre-stained protein standards. The molecular weights of the proteins in lane M are indicated on the right side of the gel in kilodaltons (kDa). The other lanes show proteins from a cleared lysate from bacteria expressing a glutathione-S-transferase (GST)-tagged protein. The proteins shown are those washed from the glutathione beads prior to elution of the GST-tagged protein.

<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>

No conflicts of interest declared.

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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24.8K Views.  University of Florida. 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°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.

Video: Pouring and Running a Protein Gel by reusing Commercial Cassettes

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 conformationally self-templating proteins that are thought to have beneficial roles in lower organisms.  Amyloids have been notoriously difficult to study due to their insolubility and structural heterogeneity.  However, resolution of amyloid polymers based on size and detergent insolubility has been made possible by Semi-Denaturing Detergent-Agarose Gel Electrophoresis (SDD-AGE).  This technique is finding widespread use for the detection and characterization of amyloid conformational variants.  Here, we demonstrate an adaptation of this technique that facilitates its use in large-scale applications, such as screens for novel prions and other amyloidogenic proteins.  The new SDD-AGE method uses capillary transfer for greater reliability and ease of use, and allows any sized gel to be accomodated.  Thus, a large number of samples, prepared from cells or purified proteins, can be processed simultaneously for the presence of SDS-insoluble conformers of tagged proteins.

SDD-AGE is a useful technique for the detection and characterization of amyloid-like polymers in cells. Here we demonstrate an adaptation that makes this technique amenable to large-scale applications.

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 (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

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 fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

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

Orthogonal Protein Purification Facilitated by a Small 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 demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

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

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

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

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

A Simple and Inexpensive Running Wheel Model for Progressive Resistance Training in Mice

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most recently, weighted-sled pulling, are highly effective at providing a hypertrophic stimulus to induce skeletal muscle adaptations. While these models have proven invaluable for skeletal muscle research, they are either invasive or involuntary and labor-intensive. Fortunately, many rodent strains voluntarily run long distances when given access to a running wheel. Loaded wheel running (LWR) models in rodents are capable of inducing adaptations commonly observed with resistance training in humans, such as increased muscle mass and fiber hypertrophy, as well as stimulation of muscle protein synthesis. However, the addition of moderate wheel load either fails to deter mice from running great distances, which is more reflective of an endurance/resistance training model, or the mice discontinue running nearly entirely due to the method of load application. Therefore, a novel high-load wheel running model (HLWR) has been developed for mice where external resistance is applied and progressively increased, enabling mice to continue running with much higher loads than previously utilized. Preliminary results from this novel HLWR model suggest it provides sufficient stimulus to induce hypertrophic adaptations over the 9 week training protocol. Herein, the specific procedures to execute this simple yet inexpensive progressive resistance-based exercise training model in mice are described.

This procedure describes a translatable progressive loaded running wheel resistance training model in mice. The primary advantage of this resistance training model is that it is entirely voluntary, thus reducing stress for the animals and the burden on the researcher.

Previously developed rodent resistance-based exercise models, including synergistic ablation, electrical stimulation, weighted-ladder climbing, and most ...

Simplifying Angiogenesis Studies with a Modified Matrix Gel Plug Assay

Modified In Vivo Matrix Gel Plug Assay for Angiogenesis Studies

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized equipment, or are hard to perform for subsequent quantitative analysis. Here we present a modified matrix gel plug assay to evaluate angiogenesis in vivo. In this protocol, vascular cells were mixed with matrix gel in the presence or absence of pro-angiogenic or anti-angiogenic reagents, and then subcutaneously injected into the back of recipient mice. After 7 days, phosphate buffer saline containing dextran-FITC is injected via the tail vein and circulated in vessels for 30 min. Matrix gel plugs are collected and embedded with tissue embedding gel, then 12 &#181;m sections are cut for fluorescence detection without staining. In this assay, dextran-FITC with high molecular weight (~150,000 Da) can be used to indicate functional vessels for detecting their length, while dextran-FITC with low molecular weight (~4,400 Da) can be used to indicate the permeability of neo-vessels. In conclusion, this protocol can provide a reliable and convenient method for the quantitative study of angiogenesis in vivo.

Author Spotlight: Investigating Angiogenesis and Vessel Permeability Through a Modified Matrix Gel Plug Assay

The method presented here can evaluate the effect of reagents on angiogenesis or vascular permeability in vivo without staining. The method uses dextran-FITC injection via the tail vein to visualize neo-vessels or vascular leakage.

Several models have been developed to investigate angiogenesis in vivo. However, most of these models are complex and expensive, require specialized ...