Fast Protein Liquid Chromatography (FPLC)
CMI FPLC resources and user guide ⬇︎
Fast Protein Liquid Chromatography (FPLC) a preparative chromatography method used for moderate pressure separation of protein samples using aqueous solvents on a variety of columns. Two of the most commonly applied FPLC methods are Size Exclusion Chromatography (SEC) and Ion-Exchange Chromatography (IEX). SEC separates by size, IEX separates by charge.
The CMI has an AKTA Pure chromatography system from Cytiva with block fraction collector.
Applications
- Size-exclusion chromatography (Gel Filtration Chromatography)
- Ion-exchange chromatography
Key Features
- Multiwavelength UV detector (up to 3 concurrent wavelengths)
- Conductivity cell to monitor solvent
- Binary (or Quad) pump for gradient elution
- Sample loop and sample pump
- 96 deep-well block fraction collection
FPLC Separation Modes
- Purification of protein by size (hydrodynamic radius).
- Useful for removing soluble aggregates and for buffer exchange.
Can be used to estimate molecular weight (from calibration with globular protein standards).
Size-Exclusion Chromatography (SEC), also known as Gel Filtration Chromatography, separates macromolecules in solution by size and shape. Size-exclusion columns are packed with fine, porous beads composed of dextran polymers, agarose, polyacrylamide, or silica. The bead pore sizes determine the size of macromolecules separated by the column. Proteins are separated by hydrodynamic radius, with large proteins eluting before small proteins. Particles that are larger than the pore size will pass through the column quickly and elute first, in the “Void peak”. The smallest molecules, including sample solvent, will elute last, because they can fully diffuse into the smallest of the pores, therefore spending the most time in the column before eluting, in the “Solvent peak.” For globular proteins, hydrodynamic radius, and molecular weight (MW) are proportional, so SEC is often used to estimate the mass and oligomeric state of protein particles by comparison to a set of globular MW standards.
- Purification of protein by charge.
- Anion-exchange columns bind negatively charged proteins.
Cation-exchange columns bind positively charged proteins.
Ion-exchange chromatography involves separation by charge. In Anion Exchange Chromatography, the resin has a positive charge allowing negatively charged proteins to bind. Proteins are negatively charged at a pH above their pI, and will typically bind to an anion exchange column under low salt conditions. Proteins that are positively charged will not bind and elute in the void volume. Negatively charge proteins can then be eluted by increasing the salt concentration. In This is often done with a gradient allowing high-resolution separation of proteins with different charges. addition to Charge, size and tertiary structure affect binding to the ion-exchange matrix. In Cation Exchange Chromatography, the matrix is negatively charged for capture of proteins that are positively charged in the loading buffer.
FPLC Supplies
- 96 deep-well blocks
- Greiner Bio-One MASTERBLOCK 96-well, 780270 (2ml)
- Porvair Sciences Microplate Deep Well, 219002 (1ml)
- User-prepared buffers compatible with chromatography method
Sample Requirements
Samples
- All samples must be BSL1-compliant (unless prior approval is requested from the CMI).
- All samples loaded onto a shared CMI column must be partially purified (by IMAC or other affinity chromatography).
- Protein samples should be in a buffer compatible with the SEC Running Buffer.
- Before sample injection, remove precipitates by centrifugation at 13,000 RPM for 10 min or filtration using 0.2 μm filter.
- Column capacity (and detector limits) determine the usable protein concentration range.
- Size Exclusion Chromatography
- 10/300 - Superdex or Superose (10 mm x 300 mm, 24 ml)
- 25 – 500 μL
- up to 10 mg
- 16/600 - Superdex (16 mm x 600 mm, 120 ml)
- < 5 ml
- up to 200 mg
- 10/300 - Superdex or Superose (10 mm x 300 mm, 24 ml)
- Ion-Exchange Chromatography
- 5/50 - Mono (Q or S) or Capto HiRes (Q or S)
- up to 5 ml with sample loop or 100+ml with sample pump
- up to 50 mg (10 mg recommended)
- 5/50 - Mono (Q or S) or Capto HiRes (Q or S)
- Size Exclusion Chromatography
Inject no more than ½ total the injection loop volume (e.g. 250 μL in a 500 μL loop)
Running Buffers/Solvents
- CMI chromatography systems are tolerant of Aqueous Solvents Only.
- CMI systems are NOT compatible with organic solvents, including acetone, acetonitrile, DMAc, DMF, DMSO, THF, TCE or with concentrated acids.
- All buffers should be filtered and degassed before use.
- Running buffer should always be chosen to be compatible with the column and the protein sample.
- SEC buffers should have a minimal osmolarity of 0.15 M, to avoid non-specific binding.
- IEX requires a low salt buffer (A) and a high salt buffer (B). High salt may be 1-2 M.
- Superdex, Superose, Capto HiRes columns are compatible with a broad range of aqueous buffers and salts, pH and additives. Common buffers include PBS, HBS, or TBS.
- Silica SEC columns cannot tolerate pH above 7.5.
- When using reducing agent seal buffer cap with parafilm to minimize oxidation over time.
- Keep an aliquot of buffer to use as baseline for quantification by UV after purification.
Prepare sufficient buffer for column equilibration and all sample runs.
Shared Columns
The CMI recommends that users purchase their own SEC columns. There will typically be a few columns available for users to borrow.