Sunday, March 7, 2010

10 Tips for Successful Sample Concentration and Buffer Exchange

10 Tips for Successful Sample Concentration and Buffer Exchange

Centrifugal devices with ultrafiltration (UF) membrane can solve common problems researchers face when working with proteins.

You’ve finally purified all the protein you need to study its structure, function, and thermal stability. Unfortunately, the protein is in a solution that you can’t use. It’s too dilute, in too large of a volume, and the buffer contains so much salt and detergent that it will adversely affect protein function or kill any cells that come near it.
Centrifugal devices with ultrafiltration (UF) membrane can solve all of these problems for proteins, nucleic acids, virus preps, and more. Harness the power of ultrafiltration membranes to separate and concentrate dissolved macromolecules, and you can prepare samples that provide reproducible, accurate data for any downstream analysis.

Particle size dictates what type of membrane-based sample preparation tool should be used
click to enlarge
Figure 1: Particle size dictates what type of membrane-based sample preparation tool should be used. (All images courtesy: Millipore Corp.)

Why should I use UF instead of dialysis or lyophilization?
UF is far gentler to solutes than non-membrane-based processes such as precipitation or lyophilization. It is easier, faster, and higher yielding than dialysis. UF is more efficient because it can simultaneously concentrate and desalt solutes. There is no phase change, which often denatures labile species, and you can perform UF at room temperature or in a cold room or refrigerated centrifuge.
Follow these tips to enhance your research with ultrafiltration.

#1 Make sure UF is the right tool for your sample.
There are many types of membrane-based sample preparation tools out there. Which one should you use? The answer depends on the size of your molecule (figure 1).

A typical centrifugal ultrafilter
click to enlarge

Figure 2: A typical centrifugal ultrafilter.

#2 Choose the right UF device for your sample.
First, select the device based on starting volume, final volume or desired concentration factor (figure 2). Centrifugal filters come in a variety of sizes, accommodating samples between 0.5 mL and 15 mL. Final volumes can vary between 15 µL and 200 µL, and concentration factors are generally between 30× and 75×.
Second, choose the membrane with an appropriate nominal molecular weight limit (NMWL), which is equivalent to the molecular weight cutoff (MWCO). You’ll get the best results with a membrane with MWCO that is 2× smaller than the molecule you wish to retain. If you have the choice between two cutoffs, pick the smaller one to start.
For most single-stranded and double-stranded nucleic acids, cutoffs between 30,000 to 100,000 give the best recoveries.

#3 Make sure your sample is chemically compatible with the centrifugal filter.
Centrifugal ultrafilters can withstand a certain pH range and a certain percentage of organic solvent. Outside of these ranges, the filter can crack, leak, or become otherwise compromised, resulting in sample loss or sample contamination.
Read the manufacturer’s recommendations provided with the filter to make sure the chemical composition of the sample doesn’t affect the filter’s performance.

Concentration polarization working against you
click to enlarge
Figure 3: Concentration polarization working against you: Buildup of solutes (blue) on the membrane prevents impurities (yellow) from passing.
#4 Avoid concentration polarization.
Concentration polarization refers to a buildup of solutes on the surface of the membrane. This prevents low molecular weight solutes from passing through the pores as they would normally do, resulting in inefficient sample concentration or desalting (figure 3).
Steps you can take to prevent concentration polarization:
• Dilute highly concentrated samples.
• Use a centrifugal filter with a vertical membrane.
• Spin at a lower speed.

#5 Actually, four tips to speed up the UF process:
• Use a filter with a vertical membrane to prevent concentration polarization.
• Choose the highest possible MWCO—up to half the molecular weight of the retained molecule.
• Remove large particles from (pre-clarify) the sample by syringe microfiltration or centrifugal microfiltration.
• Read the filter manufacturer’s guidelines to decide exactly how long to centrifuge your sample and whether dividing the time into multiple centrifugation steps can speed up the concentration process.

#6 Keep your molecule biologically active for best assay results.
UF is conducive to preserving biological activity of solutes, because it does not typically change the salt concentration of the solution. However, you should:
• Avoid spinning to dryness. Using a filter with a dead stop enables concentration in a single spin and concentration of multiple samples in parallel without calibrating spin times.
• Ensure that the molecule is soluble at the final concentration.
• Use a refrigerated centrifuge for temperature-sensitive molecules.
• When concentrating viruses, test activity at various titers and stop concentrating further when activity shows any signs of diminishing.

Use inverted spin for consistent recovery of low volume samples
click to enlarge

Figure 4: Use inverted spin for consistent recovery of low volume samples with minimal pipetting errors.
#7 Maximize recovery of your molecule of interest—recover 90% of your sample or more.
• Use a membrane with an appropriate MWCO.
• Use a centrifugal filter with a vertical membrane.
• Choose a low-binding membrane to minimize nonspecific binding of solutes (e.g., regenerated cellulose acetate membrane with polypropylene housing).
• Use a reliable centrifugal filter that has been validated to function without leakage or breakage. See Tip #2 for advice on chemical compatibility.
• Choose the correct rotor and centrifugation speed for the filter by following the manufacturer’s directions. The wrong choices here could also structurally compromise the filter, causing lost sample.
• Avoid spinning to dryness. For peace of mind, use a filter with a dead stop (volume past which sample cannot be further concentrated).
• For consistent recovery of low volume samples with minimal pipetting errors, invert the device after concentration and spin again into a new collection tube (see figure 4).

click to enlarge

Figure 5: How to use a centrifugal filter to concentrate a protein 10× while also gradually reducing the salt concentration by 9×.
#8 Use gradual desalting for sensitive macromolecules.
Because some macromolecules can lose activity or proper structure upon extreme changes of buffer conditions, change buffer conditions gradually or use diafiltration. Diafiltration involves removing microsolutes by adding solvent to the sample being filtered at the same time that ultrafiltration is being applied.
Figure 5 shows an example of how you can use a centrifugal filter to concentrate a protein 10× while also gradually reducing the salt concentration by 9×.

#9 Dilute samples with concentrated detergents in the buffer.
Dilute the sample so the detergent concentration is low enough that micelles (large, organized detergent aggregates) do not form. If the sample contains micelles, UF will concentrate the detergent even further. Different detergents have different critical micelle concentrations.

#10 Tell all your colleagues about the ease and effectiveness of UF.
The more people around you that use UF, the more tips you can share on optimizing UF. Here’s a partial list of UF applications:
• Protein applications, including concentration and buffer exchange
• Concentrating chromatography fractions
• Concentration antibodies
• Desalting any protein sample, instead of dialysis
• Removing detergents
• Nucleic acids
• PCR clean-up and primer removal, purification of DNA sequencing reactions
• Genomic DNA concentration and desalting
• Clinical applications
• Deproteination of clinical samples for drug discovery & development
• Concentration of biological samples (urine, cerebrospinal fluid, blood) for analysis
• Measuring the levels of unbound drugs, heavy metals, or other soluble molecules (such as hormones used for doping)
• Separating free from bound metabolites in serum samples
• Viruses used for gene transduction
• Concentration by UF is efficient, but it is difficult to separate soluble proteins from viruses in cell culture media by UF. Use UF to concentrate and desalt virus preparations only.

What’s next?
Now that you have your native, folded, active molecule in a usable volume and concentration, your next assay will more likely be a published figure, instead of a “Troubleshooting” slide in your next group meeting presentation.

About the Authors
Lisa Morrill leads the direction of technology development at Millipore including sample concentration, purification, and immunodetection portfolios.
Sandrine Mercier integrates development and marketing for the Amicon Ultra centrifugal filters at Millipore.

This article was published in Bioscience Technology magazine: Vol. 32, No. 10, October, 2009, pp. 14-19.


Post a Comment

Design by sudhanshu. Converted To Web By SUDHANSHU RATNA THAKUR