The Pall Life Sciences Microsep™ Centrifugal Devices are an easy way to purify, desalt and concentrate biological samples. The manual states that 3.5 ml of sample can be concentrated 100 fold in 30-90 minutes. The Microsep™ Centrifugal Devices use an Omega™ ultrafiltration membrane to trap biomolecules larger than the specified molecular weight cut-off. The lower molecular weight molecules and solvent pass through the membrane. There is a wide range of molecular weight cut-off Microsep™ devices so they can be used to concentrate small peptides, oligonucleotides, nucleic acids, enzymes, antibodies, microbes and macromolecules. The Omega™ membrane does not contain a rubber O-ring: an advantage since rubber can cause non-specific adsorption and contamination. The Microsep™ Centrifugal Devices use centrifugal force at an angle to prevent the build-up of protein on the membrane itself, allowing the biomolecules to collect on the plastic ring surrounding the membrane. This also prevents filtration to dryness and allows for a ‘dead-stop’ or point when nothing more will pass through the membrane. The membrane can also be used without pre-rinsing, unless glycerine and sodium azide will interfere with the downstream assay as the membrane is coated in the glycerine and sodium azide.
The Microsep™ Centrifugal Devices are very easy to use after reading the manual completely. Determining which device to use requires knowledge of the molecule you are trying to concentrate. A table is provided in the manual to help you decide which one to use and they also recommend using a cutoff 3-6 times less than the molecular weight of your molecule. For example, if your protein is 150K, a 30K or 50K Microsep™ is what they recommend. If DNA is being concentrated, multiply the number of bases by 340 for single-stranded and by 680 for double-stranded DNA and then select the device that is 3-6 times less than that molecular weight. For example, a 2 kb double stranded DNA fragment (2000X 680= 1360K Daltons) should be purified using a 300K Microsep™ device. The devices are also color coded which helps to keep them organized.
Once the correct cut-off size has been determined, up to 3.5 ml can be loaded in the sample reservoir. The device is then put in a fixed angle centrifuge and spun at 3,000-7500 X g for 30-90 minutes. There are also tables within the manual to give more specific speeds and times. The molecular weight cutoff of the device as well as the solute concentration alters the speed and time of centrifugation. Once the centrifuge has stopped, the concentrated sample can be removed with a pipette and stored in their provided concentrate cup and the filtrate can be capped and stored as well.
I used these devices to concentrate an unknown small peptide after affinity chromatography. I did find that glycerol effected the dead stop time and would not allow complete concentration. I discussed this with customer service a few times and they were very helpful. They recommended using a larger cutoff device first to do an initial concentration and then adding the filtrate to a smaller cutoff device. This leads to more complete concentration by separating the peptides in to smaller fractions. I wanted to further concentrate my target peptide which was accomplished by adding buffer to the reservoir of the column that contained the target peptide size and respinning in the same column. Although I did not complete the process in 90 minutes, I had started with more than 3.5 ml, so multiple spins had to be done.
Overall, the Microsep™ Centrifugal Devices were easy to use and the manual was detailed and informative. These devices are said to replace dialysis, chemical precipitation and lyophylization when concentrating samples for electrophoresis, buffer exchange or salt removal of chromatography, de-proteinizing serum or urine for HPLC, isolation of biomolecules, separating primers and/or purifying hybridization probes. Some factors that may affect performance are protein concentration, protein: protein interactions, temperature, ionic concentration and the size of the molecule. The devices work best with 1 mg/ml or less of protein and colder temperatures lead to slower flow rates.
Overall, these devices are a great tool for concentrating molecules. I only have experience with peptide concentration, but this worked well for my application. The manual is detailed and can be confusing, but once deciphered, the concept is quite easy.
Katrina Stumpf
Senior Lab Specialist
McGuire VA medical Center
Division of Infectious Diseases