Description
Guidelines for PolyCAT A
Structure of Product Description:
Column Name- Particle diameter(um)- Pore Size( A) Column length(mm)x ID(mm)/Art No.
Capillary Name-Particle diameter(um)- Pore Size( A) Column length(mm)x ID(mm)
Bulk Material Name -Particle diameter(um)- Pore Size( A)/Art No.
Uniqueness of PolyCAT A
PolyCAT A™ is made by attaching covalently poly(aspartic acid) to silica. Proteins elute from this polypeptide coating in sharp peaks with little tailing. Moreover, recovery and binding capacity is high.
Overview of Applications
1) Protein variants involving deamidation, PEGylation, determining position of attachment, desialylation and many more. It is widely used by the biotechnology industry.
2) Hemoglobin variant analysis by clinical chemistry labs.
3) Specifically for Proteins with pI above 6.0 (5.0 in special cases).
4) Monoclonal antibodies.
5) Histones.
Properties of PolyCAT A
PolyCAT A™ is a weak cation-exchange (WCX) material. It is used at pH values above 4. A gradient to unbuffered acetic acid will uncharged PolyCAT A™, permitting the elution of proteins in a volatile solvent. See our poster on this subject.
Peptides can be run on PolyCAT A™ if they contain at least two excess positive charges above pH 4. More weakly basic peptides, such as tryptic fragments, are not reliably retained. Instead use PolySULFOETHYL A™ at pH = 2.7 – 3.0
For proteins larger than 20 KDa, we recommend the use of pore diameters particles. Use at least 1000 Å Porediameter for optimal selectivity and efficiency. Our 3-µm material with 1000- or 1500-Å pores is the finest cation-exchanger available for protein separations.
How professionals work with PolyCAT A
Initial Use:
PolyCAT A™ is a silica-based material with a bonded coating of polyaspartic acid. It is a weak cation-exchange (WCX) material. Columns are shipped in methanol. Flush new columns with at least 15 column volumes of water (30 ml for a 200 x 4.6-mm), then condition with a salt solution prior to initial use. A good conditioning solution is 40 mM EDTA.2Na (filtered, but pH not adjusted) at a low flow rate for 20-24 hours.
New HPLC columns sometimes absorb small quantities of proteins or phosphorylated peptides in a nonspecific manner. The sintered metal frits have been implicated in this. Eluting the column for 20-24 hr. at a low flow rate with 40mM EDTA.2Na usually solves the problem. This passivates all metal surfaces in the HPLC system, as well as the column [CAUTION: This treatment can affect the integrity of the frits in some cases, and should probably be avoided with columns packed with 3-µm material. In some cases this has also caused the collapse of 5-µm, 200-Å column packings]. Alternatively, after flushing with water, condition the column for 2 hours with 0.2 M NaH2PO4 + 0.3 M sodium acetate. This solution conditions the coating but does not passivate metal surfaces.
Routine Use
Proteins can be eluted from PolyCAT A™ columns with salt and/or pH gradients. The most useful range for cation- exchange of proteins is pH 6-7. Phosphate and Bis-tris are good buffers in this range. The higher the pH, the weaker the retention. Avoid prolonged exposure to a pH above 8.
For weakly basic peptides or for cation-exchange below pH 4, use PolySULFOETHYL A™, our strong cation-exchange (SCX) material.
Use ambient temperature (20-25°C), as this polypeptide-based coating is more sensitive to elevated temperatures than are other materials. Filter mobile phases and samples before use. Failure to do so may cause the inlet frit to plug. This frit can be replaced. At the beginning of the day, flush the column with 15 column volumes of the high-salt buffer before equilibration with the low-salt buffer. At the end of the day, flush the column with 15 column volumes of water and plug the ends.
Loading Capacity:
The loading capacity of a 4.6mm ID column is about 4 mg of protein/injection, depending on the strength of the protein’s binding to the support.
Storage:
1) Overnight: 100% mobile phase A. 2) Several days: Store in water. 3) Longer periods: Store in water in the refrigerator, with the ends plugged. ACN can be added to the storage solvent (e.g., ACN:Water = 80:20) to retard microbial growth.
Column maintenance:
After every 250 runs, invert the column and run it backwards overnight, at a low flow rate, with 40 mM EDTA.2Na. Continue using the column in this inverted direction for the next 250 samples, then repeat this treatment. If possible, open the inlet and fill in any voids with bulk PolyCAT A™ after running 500 samples.
