Name: using the gelfree 8100 fractionation system for molecular weight-based fractionation with liquid phase recovery
Uploaded: 2009-12-03T16:30:00
Duration: 7 min 57 s
Description: Watch this Scientific Journal Video about Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery at JoVE.com

1. Sample Protein Preparation
<ol>
 <li>In a 500 &mu;l microfuge tube, combine the following:</li>
</ol>
<table border="1">
<tbody>
 <tr>
 <td>Reagent</td>
 <td>Volume to add:</td>
 </tr>
 <tr>
 <td>*Protein sample (up to 1 mg)</td>
 <td>Up to 112 &mu;L</td>
 </tr>
 <tr>
 <td>Sample Buffer (5X) provided in cartridge kit </td>
 <td>30 &mu;L</td>
 </tr>
 <tr>
 <td>DTT 1M</td>
 <td>8&mu;L</td>
 </tr>
 <tr>
 <td>18 &Omega; H2O</td>
 <td>x &mu;L to a total of sample volume of 150&mu;L**</td>
 </tr>
</tbody>
</table>
<ol start="2">
 <li>Denature the samples at 95&deg;C for 5 minutes. Cool to room temperature.</li>
</ol>
*The total amount of protein to be separated will depend on the complexity of the sample. For more complex samples, such as tissue homogenate, 150-200 &mu;g per channel is recommended.
**If the sample volume is less than 112 &mu;L, add 18 &Omega; H2O to bring the final volume to 150 &mu;L.
2. Loading the GELFREE Cartridge
<ol>
 <li>Remove the GELFREE 8100 Cartridge from the foil pouch. </li>
 <li>Remove and discard the plate sealer. </li>
 <li>Remove the storage buffer from the cartridge compartments using a pipette. If all 8 chambers of the cartridge will be used, the cartridge can be inverted to drain the storage buffer from the compartments. </li>
 <li>Prepare the cartridge for loading by adding GELFREE Running Buffer to each of the cartridge chambers as follows:</li>
</ol>
<table border="1">
<tbody>
 <tr>
 <td>GELFREE Cartridge Chamber</td>
 <td>Volume of GELFREE Running Buffer to add</td>
 </tr>
 <tr>
 <td>Anode buffer reservoir</td>
 <td>8 mL</td>
 </tr>
 <tr>
 <td>Cathode buffer reservoir</td>
 <td>6 mL</td>
 </tr>
 <tr>
 <td>Collection chambers</td>
 <td>100 &mu;L each</td>
 </tr>
 </tbody>
</table>
<ol start="5">
 <li>Remove and discard any buffer from the sample loading chamber.</li>
 <li>Load the 150 &mu;L samples into the loading chambers using an 8-channel pipettor.</li>
 <li>Place the loaded cartridge into the GELFREE 8100 Fractionation Station. Lower the electrode arrays and close the lid.</li>
</ol>
3. Running the cartridge
 <ol>
 <li>Using the instrument s touch screen display, press the method button to view the list of pre-programmed methods. Each of the pre-programmed methods consists of set voltages and time-based pauses designed to resolve proteins within a specific range of molecular weights. The GELFREE 8100 will automatically pause for fraction collection at the intervals specified in the method.</li>
 <li>Once the appropriate method has been identified, press the retrieve button, and enter the number of the desired pre-programmed method using the on-screen keypad. Press ok, then press the apply button. The method that has been applied will appear at the bottom of the main screen. </li>
 <li>To select the channels (number of samples) that will be used in the run, press the channel button on the main screen. To select all eight channels, press select all. Then, press done to return to the main screen. </li>
 <li>Ensure that the safety lid is closed and that the indicator light is green at the bottom of the screen. </li>
 <li>To begin the protein separation, press start. The Fractionation Station will automatically pause when it is time to collect fractions. Note: While the system is running, the circles shown in the box on the left side of the screen depict the status of each channel. A green circle indicates that the channel is active, a yellow circle indicates that the instrument has paused, and a red circle indicates that the channel has failed. The applied voltages and currents are displayed to the right of the channel status circles. Fractions should only be removed when the status circles have turned yellow and a text alert appears on the instrument s screen.</li>
 <li>To remove fractions, open the lid of the instrument and use an 8-channel pipette to withdraw 150 &mu;L from each of the collection chambers. </li>
 <li>Once the fractions have been removed, wash the collection chamber twice with GELFREE Running Buffer by adding 100 &mu;l per channel and pipetting up and down twice. </li>
 <li>After washing, add 100uL of GELFREE Running Buffer back into the collection chambers, close the lid, and press the resume button. </li>
 <li>The GELFREE 8100 will run until the next time interval. The total time remaining in the experiment is shown on the right hand side of the screen. To toggle between time remaining, time elapsed, and time to pause, press the button above the box displaying time.</li>
 <li>Once all of the fractions have been collected, they can immediately be used for further downstream preparation, such as isoelectric focusing or gel electrophoresis, or for analysis using liquid chromatography mass spectrometry or immunoaffinity.</li>
</ol>
4. Representative Results
A 200 &mu;g of bovine liver homogenate was partitioned into 12 fractions each, using the GELFREE Fractionation System. 6 &mu;L of each fraction was run on a standard 1D SDS PAGE gel and silver stained. As shown in Figure 1, the protein sample was fractionated into 12 distinct fractions, spanning the molecular weight range from 3.5kDa to 150kDa. A molecular weight standard (ColorBurst, Sigma) are also shown in lanes 1 and 14. The gel shown here highlights the high-recovery rate achieved using the GELFREE 8100 system for protein fractionation. The gel shown here represents 4% of the protein recovered from 200 &mu;g.
<img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="Figure 1" border="0" /> Figure 1. Proteins from bovine liver homogenate separated using the GELFREE Fractionation system. 6 &mu;L of each 150 &mu;L fraction was loaded and run on a 1D SDS PAGE gel and silver stained. A molecular weight (MW) standard was also loaded as indicated. Please <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">click here</a> to see a larger version of figure 1.

<ol>
	<li>Tran, J. C., Doucette, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Size+Separation+of+Intact+Proteins+in+Solution+Phase+for+Mass+Spectrometry.">Multiplexed Size Separation of Intact Proteins in Solution Phase for Mass Spectrometry.</a> Anal Chem.. , (2009).</li></ol>

The authors of this article are employed by Protein Discovery that produces reagents and instruments used in this article.

The GELFREE 8100 Fractionation System provides a quick, simple and reproducible method for partitioning complex protein samples into discrete molecular weight-based fractions. This novel method of fractionation eliminates the need for band or spot cutting, making it possible to isolate proteins over a broad mass range in 90 minutes. The ability to load relatively large amounts of sample and recover fractions in liquid phase is particularly useful in the isolation and analysis of low-abundance proteins, which are typically more difficult to detect. The resulting fractions can then be used in a wide array of applications, such as LC-MS, MALDI-TOF, and immunoblotting.

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.

Watch this Scientific Journal Video about Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery at JoVE.com

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

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 ...

using-gelfree-8100-fractionation-system-for-molecular-weight-based

Research

JoVE Journal

Biology

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Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

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In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

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Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

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Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

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In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

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Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

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Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

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Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

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This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...