Where to buy spin filters for FASP? Millipore has recently ceased production of the Microcon units (however, they are reported restarting production; Cat. MRCF0R030). Filters that are similar to the 0.5 ml Microcons can now be purchased from Sartorius-Stedim (Cat. VN01H22). The alternative are Vivacon 500 units with nominal cut offs of 30,000 or 50,000. We compared them to the Microcons and have obtained similar results. In our hands, the best yield can be achieved using Vivacon and Microcon with flat bottoms (because the membrane is oriented perpendicularly to the centrifugal force). Units with V-shaped oriented filters (Amicon Ultra-0.5) usually provide lower yields due to their higher dead-volume.
What is the expected yield obtained by the FASP method? The typical yields obtained by FASP are about 50% of the applied protein sample. This is true over a wide range of protein amounts from few micrograms (processed in 0.5 ml units) up to 20 milligrams (15 ml units). The “50% value” has been criticized as being low, but to our knowledge FASP is unique in allowing precise monitoring of the digest yield. Neither the in-gel nor the in-solution (urea/thiourea) methods enable analysis of the efficiency of digestion. For this reason, it is not possible to directly judge the efficiency of FASP against these established methods – however, we think it compares quite favorably.
What are the sample loading limits for the 0.5 ml units? We have tested the efficiency of FASP performed in 0.5 ml filtration units in the range of 0.1-400 microgram of total protein. Using different cell and tissue lysates we observed yields of peptides corresponding to 50% of the applied sample.
How does one process large sample amounts? For total protein amounts higher than 0.4 mg, concentrators with capacities of 4 or 15 ml should be employed. The 4 ml and 15 ml Amicon Ultra Units are routinely used for processing of 5 and 25 mg total protein.
Why do we recommend filters with the 30,000 cut-off? Denatured proteins remain unfolded in urea. Due to their large Stokes’ radii, they are retained in the concentrators during the buffer exchange procedure. Importantly, peptides with sizes up to 5,000 can be collected using 30k filters. In contrast, the units with cut offs of 3,000 or 10,000 filters retain larger peptides. In addition, the time required for concentration using the 3,000 and 10,000 filters is several folds longer when compared to 30,000 filters. For reference see: Anal. Biochem. 410, 307 (2011)
What does it mean when the buffer is exchanged too slow or too fast? Typically 10 – 15 minutes at 14,000 × g are required for concentration of the 0.2 ml 8 M urea solution into about 10 µl in a 0.5 ml Microcon or Vivacon unit. Applying too high amounts of protein lysate can result in clogging of the filters. This leads to increase of the required centrifugation times. If the urea solution passes through the filter too quickly (a minute or less), this is typically a sign of a membrane perforation. In our experience, some batches of Microcon filters have contained many units that only pass the sample instead of concentrating it).
How does one determine the initial protein amount in the SDS lysate and how does one measure the amount of obtained peptides? Determination of total protein amount can be difficult when samples are diluted and contain chaotropic and micelle forming reagents such as urea and detergents. Under such conditions colorimetric assay typically tend to overestimate the protein content. We routinely use fluorometry to determine tryptophan concentration in the samples using tryptophan as a standard. This fluorometric assay allows determination of protein amount in the low microgram range. The fluorometric assay can be used both for determination of the protein in the sample and of the peptide obtained by FASP. The peptide concentration can also be measured directly by UV spectrometry.
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