Gelfree 8100 Fractionation System FAQ
Basics
What does the Gelfree 8100 Fractionation System do?
The Gelfree 8100 System fractionates complex protein mixtures into liquid-phase molecular fractions. Its like being able to cut out any number of bands in a 1-D and extract them into solution, but without slab gels and cutting, and with much higher recoveries. And, proteins are recovered intact, so you can analyze post-translational modifications or tie the intact protein molecular weight to the identified peptide in shot gun experiments.
Is protein recovery affected by pI or hydrophobicity?
No. Unlike chromatographic methods, molecular weight fractionations using the Gelfree 8100 System are unbiased by protein pI or hydrophobic character.
What is the buffer composition?
Gelfree cartridges use a specially designed HEPES-SDS buffer system.
What is the pH of the running buffer?
The pH of the running buffer is 7.8.
Do the sample and running buffers contain SDS?
Both the sample buffer and the running buffer contain SDS.
What is the gel?
The Gelfree 8100 System uses proprietary polyacrylamide gels that have been specially designed for maximum resolution, reproducibility, and robustness.
How many times can you use a Gelfree cartridge?
Each of the eight channels in a cartridge is an independent system which can be used once. If you have one or two samples for fractionation, you can process just those samples using the Gelfree 8100 System without using up a whole cartridge. The unused channels will still be available for use at another time.
Features
What is the throughput of the system?
A typical run time is two to three hours to fractionate up to eight samples simultaneously over the entire mass range of 3.5 kDa to 300 kDa.
How long does it take to fractionate proteins?
If the only proteins of interest are of low molecular weight, a fractionation can be finished in just a few minutes. If multiple molecular weight fractions are being collected, or if high molecular weight proteins are targeted for fractionation, run times are longer.
What is the mass range for protein fractionation?
The Gelfree 8100 System enables liquid-phase protein fraction collection from 3.5 kDa to 300 kDa.
How much protein is recovered?
Total recovery will depend on the protein of interest. For albumin (MW 66 kDa), recovery was determined to be 64%.
What is the reproducibility for the quantity of recovered proteins?
The Gelfree 8100 System is highly reproducible. For example, the CV for the amount of albumin recovered through the Gelfree system was determined to be 8.4% (n=8).
What is the typical mass resolution?
The mass resolution is typically 10% of the range at the molecular weight of the fraction. At 50 kDa, the range is approximately 7-10 kDa. For optimum resolution within a particular range, select a cartridge for which the range of interest is at or near the center of the optimum range specified for the cartridge. In other words, select a cartridge where the range of interest is near fraction 6 when using the method outlined on the Quick Reference Card.
What is the advantage of the Gelfree technology over size exclusion chromatography?
The Gelfree system provides a wider mass range (from 3.5 kDa to 300 kDa), better yield, and, in many instances, better resolution. With multiple-channel, parallel throughput, the Gelfree system provides faster results and does not require an LC column, so you dont have to dedicate an LC system or switch it back and forth for different applications.
Sample Loading
How do I prepare cells for protein fractionation on the Gelfree System?
Ideally, use serum-free media for cell culture. This will help avoid contamination of the sample with albumin.
For protein extraction from cells, we recommend the UPX Universal Protein Extraction Kit. This product is compatible with the Gelfree System and frankly outperforms other commercially available lysis buffers by a wide margin. For starting amounts, we do not recommend a specific number cells; rather, we have based our use instructions on a wet pellet mass. This measure is generally applicable to a variety of cell types. As you gain experience with the cell types you are fractionating, you can substitute another starting material measurement method if you desire.
We recommend also that you use a protease inhibitor mixture, such as our Proteoloc Protease Inhibitor Cocktail, to protect extracted proteins from degradation by endogenous proteases.
For quantification, we recommend the Proteoquant Proteome Quantification Assay Kit because it is compatible with reducing agents and detergent.
Can I simply add Acetate Sample Buffer (5X) and reducing agent to my sample, or do I need to lyse and desalt my sample prior to fractionation on the Gelfree System?
Better results are obtained if the samples are lysed and desalted.
For sample lysis, we recommend the UPX Universal Protein Extraction Kit. The UPX Kit is compatible with the Gelfree System and delivers an abundance of unbiased protein for fractionation and analysis.
Desalting samples decreases fractionation variability between sample types. The voltage drop across the sample loading chamber is reduced by saltier, more conductive samples. The corresponding electric field through the sample reduces the loading rates of the ions into the gel. While desalting might not be required for successful fraction, uniform sample conductivities will produce more reproducible results.
What is an acceptable salt concentration for a sample prepared for fractionation on the Gelfree System?
For fractionation to proceed similarly to those described on the Quick Reference Cards, sample conductivity should be < 2 mS/cm. For sodium chloride, this translates to a salt concentration of approximately 20 mM. Desalting samples will ensure uniform conductivities and produce more reproducible results.
If your sample is already at a low conductivity, there is no benefit to desalting.
If the sample conductivity is > 2 mS/cm, desalting it will reduce conductivity to < 1 mS/cm.
If you choose not to desalt a sample with a conductivity > 2 mS/cm, be aware that the sample elution time will be increased. Samples can be run in this manner, but one should not expect the fraction times to correspond with those on the Quick Reference Card.
What is the loading volume?
The sample loading volume is 1 – 112 μL per channel. Total loading volume is 150 μL per channel.
What is a typical sample loading amount (μg) for one channel?
If a sample contains only a few protein components, the loading amount should be no less than 1 μg per channel.
For complex samples, typical loading amounts range up to 500 μg total protein per channel.
The relationship between excess protein loading and fractionation can be visualized by deliberately loading a large amount of a large, purified protein, and analyzing the resulting fractions on a 1D gel.
What happens if you overload the sample loading chamber with too much protein?
The resolution of the separation will deteriorate. The maximum loading amount depends on the sample, but we generally suggest loading up to 250 μg of total protein per channel. To fractionate 2 mg of total protein, you’ll get the best results by loading 250 μg of protein into each of eight channels.
Can sucrose or glycerol be present in a sample prepared for fractionation on the Gelfree System?
If the concentration of sucrose or glycerol is sufficiently low, and the sample does not sink to the bottom of the sample loading chamber, then it is acceptable.
Can I use a sample buffer that is typically used for 1D SDS-PAGE?
No. Unlike typical SDS-PAGE sample buffers, Gelfree sample buffer does not contain components that increase sample density. If a sample containing such components, such as sucrose or glycerol, is loaded on the Gelfree System, it will sink to the bottom of the Gelfree sample loading chamber. During the Gelfree run, proteins will enter the stacking gel incorrectly and migrate along the bottom of the Gelfree Cartridge channel, rather than migrating uniformly through the entire cross-sectional area of the channel. Such improper cartridge channel entry and migration will adversely affect migration times and fractionation quality.
Operation
How do I run fewer than eight channels and preserve the remaining channels for later use?
Carefully peel off the plate sealer. For the channels to be used, follow the protocol outlined in the User Manual. For the remaining unused channels, the storage buffer should be left in place and those channels should not be energized during the run. In other words, de-select the appropriate channels on the Channels Page. At the conclusion of the experiment, empty the running buffer from the used channel(s) and replace the plate sealer. Store at room temperature.
Can you apply different fractionation methods to each lane?
No. The Gelfree System is designed to run one method at a time.
If voltage is applied to the wrong lane, will that lane become unusable?
If the channel that was energized contained storage buffer, running it for a short time should be OK. However, if the reservoirs contained running buffer, the trailing ion will have entered the gel ahead of the sample, making the lane unusable. Similarly, storing running buffer in an unused lane makes the lane unusable, as the buffer diffuses quickly into the gel.
What is the maximum recommended voltage?
The recommended range is 50 – 100V. Exceeding this range can introduce adverse temperature effects that compromise reproducibility.
Do I really need to wash the sample loading chambers at the first pause?
Our recommendation is to wash the sample loading chambers because there may be residual proteins adsorbed to the walls. This effect has been observed after loading a sample containing a high-abundance protein. If a small amount of highly abundant protein enters the gel after the sample has loaded in Step 1, a 1D gel will show that band repeated across several fractions. Although it may never become an issue for many users, washing the sample loading chambers will minimize this potential problem. We recommend washing twice with a 150 μL volume. After the wash steps, add 150 μL of running buffer to the sample loading chamber, and 2 mL of running buffer to the cathode reservoir, before resuming the method.
Fraction Recovery
How many fractions can you collect per channel?
As many as you want. You can collect a single molecular weight fraction containing your protein of interest, or partition your protein mixture into dozens of fractions.
What is the volume of eluted fractions?
The volume of each eluted fraction is 150-200 μL.
What is the minimum collection volume?
150 μL is the minimum volume. If a lower volume is desired, use a vacuum centrifuge to concentrate the protein. This will also increase the concentration of Tris and SDS.
Do high molecular weight proteins come out in more dilute fractions?
No. The Gelfree 8100 System is designed specifically to limit high molecular weight protein diffusion, so the system can efficiently fractionate proteins up to 300 kDa.
What happens to proteins above the high MW specification of the cartridge?
They remain in the resolving gel.
What happens to proteins below 3.5 kDa?
They are not retained in the sample collection chamber and migrate through to the anode reservoir.
What percentage of SDS is contained in the fractions?
After the first fraction, the SDS concentration should be approximately 0.1%. The first fraction may contain slightly more SDS due to the additional SDS contribution from the sample buffer.
How should fractionated samples be stored?
Fractions may be stored overnight at 4 °C. If you need to store your fractions for more than one day prior to analysis, keep them frozen at -80 °C until you are ready to use them.
Tips
For proteins prepared from crude samples by TCA precipitation, is there any recommended protocol for sample dissolution and preparation prior to fractionation on the Gelfree System?
If you are fractionating the entire pellet, you can simply add 30 μL of Acetate Sample Buffer, 8 μL 1M DTT reducing agent, and 112 μL water. If only a portion of the pellet will be used, dissolve it in 10 mM Tris acetate buffer at neutral pH. Then add 30 μL of Acetate Sample Buffer, 8 μL of 1M DTT reducing agent, your sample, and water to 150 μL.
How can I get rid of the SDS?
Numerous high recovery methods exist to remove SDS. We have critically compared the various methods and techniques and have found spin columns from Thermo Pierce (#87777) to provide the highest recovery and ease-of use.
How can I get rid of salt?
Variations in sample salt concentration will affect protein mobility and reduce system reproducibility. Protein Discovery recommends using Pierce Zeba Spin Desalting Columns, 7K MWCO (#89882) to eliminate this issue.
Once I have collected the protein fractions, what can I do to process those samples?
If you need the intact protein for top-down analysis or for some other purpose, and you wish to clean up the SDS, we recommend the Pierce Detergent Removal Spin Column (Part #87777). Otherwise, we recommend the FASP Protein Digestion Kit.
What is the protocol for visualizing fractions produced by the Gelfree System on a 1D SDS-PAGE gel?
To produce the gel images such as those appearing on the Gelfree Cartridge Quick Reference Cards, mix a 5 μL aliquot of the Gelfree fraction you wish to analyze with 8 μL water, 5 μL LDS sample buffer (Invitrogen, NP0007), and 2 μL 1M DTT. Heat for 10 minutes at 50 °C, then load 10 μL of the heated solution onto a 15-lane gel. For fractions produced using the Gelfree 12% Cartridge Kit, use a 4-12% Tris-glycine gel (Invitrogen, EC60355BOX). For fractions produced using other Gelfree Cartridge Kits, use a 10-20% Tris-glycine gel (Invitrogen EC61355BOX). Run the gel at 120V for approximately 2 hours, then visualize proteins by silver staining.
How should Gelfree Cartridges be stored?
Gelfree Cartridges are packed room temperature and are shipped in an insulated container to avoid exposure to extreme temperatures during transit. Store cartridges at room temperature. Do not refrigerate or freeze them.
Troubleshooting
Why is one lane running more slowly than the others?
Occasionally a bubble may become trapped against the membrane separating the sample loading chamber from the gel. One can clearly identify a channel that has been occluded by a bubble because the electrical current will be much lower than the surrounding channels. When you add running buffer to the reservoirs, do so slowly so as to minimize the chances of producing a bubble. Inspect the membranes in each active channel prior to your run. If any bubbles are evident, gently free them from the membrane with your pipettor.
How can I reduce high-abundance protein carryover?
High-abundance proteins should not appear in multiple fractions. If you see such carryover, this effect can be minimized. Empty and wash the sample loading chambers twice with 150 μL running buffer at the first pause then add 2 mL of running buffer to the cathode reservoirs. This removes high-abundance proteins which may have associated with the walls of the sample loading chambers.