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GENERAL PURIFICATION OF PROTEINS
WITH PDC USING PDC KIT
- If the structure of the protein to be purified is known and if it contains one, two, three or more histidine residues, the PDC KIT can be used for a primary screening.
- Our PDC KIT can also be used as a group specific adsorbent that will separate proteins with accessible cysteine/histidine containing motifs from those that do not.
APPLICATIONS
Example 1 : with PDC-Sepharose CL-4B Kit (PDC-4S KIT)
By kind permission of the author, Dr P.A. Flamée, Laboratoire de Génie génétique et de Biologie moléculaire, Institut de Biochimie B6, Sart Tilman 4000 Liège (Belgium).
Sample volume that was loaded onto each column: 10 ml of crude clarified lysate of Mutated triosephosphate isomerase (Mutated timase) from E. Coli, containing 8 histidine residues (5 accessible).
SDS-PAGE 12% in the presence of beta-mercaptoethanol
(15 µl of sample - stained with Coomassie blue)
Panel B (Ni-PDC) |
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Panel B Fractions 1,2,3 & 4: washing with K2PO4 20mM pH 7.5, NaCl 50mM. Fractions 10-11: fractions eluted at pH 5.7. Fractions 15-17: mutated timase (MW 27,000 daltons) eluted at pH 5.0. M: Markers 14,400; 21,500; 31,000; 45,000; 66,200 & 97,400 daltons. Conclusion: Ni-PDC allows the purification of mutated timase in a single step with a recovery of 15 mg of protein per ml of wet gel (Panel B, lane 15, 16 & 17). Panels A, C & D (Cu-PDC, Zn-PDC & Co-PDC) are not shown. |
Example 2: alpha2-Macroglobulin purification
The PDC KIT allows the purification of alpha2-Macroglobulin in a single step with the purity of approx. 90% (w/w) as follows:
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Gel volume: 25 ml Ni2+PDC- Sepharose CL-4B. Sample: 100 ml human serum. Binding buffer: K2HPO4 50mM, NaCl 0.15M pH 8.0. Washing buffer: 1. K2HPO4 50mM, NaCl 0.15M pH 8.0. / 2. K2HPO4 50mM, NaCl 0.15M pH 6.0. Elution buffer: AcONa 50mM pH 3.3.  |
| Purity of alpha2-M (containing 38 histidine residues) was determined to be approx. 90% by SDS-PAGE in the presence of beta-mercaptoethanol and its activity by immunodiffusion on agarose gel. |
Example 3: Mouse monoclonal IgG purification
Ni2+-PDC-Matrix is also an excellent affinity support for the purification of Mouse monoclonal IgG1 with a recovery greater than 90% and the degree of purity greater than 90% (w/w).
Results not shown.
Sample: Clarified Ascite fluids.
Binding buffer: K2HPO4 50mM, NaCl 0.15M pH 8.0.
Washing buffer: K2HPO4 50mM, NaCl 0.15M pH 8.0.
Elution buffer: K2HPO4 50mM, NaCl 0.15M pH 6.0.
PROTOCOL FOR FIRST ATTEMPTS AT GENERAL PURIFICATION OF PROTEINS WITH PDC USING PDC KIT
1. Selection of the most appropriate Metal chelate column in a primary screening with PDC KIT
The selection of the most appropriate column can be realized in the same manner as for 6x His-tagged proteins.
2. Optimization of purification of proteins of interest with PDC-Matrix
Once the most appropriate metal chelate column is selected, the optimization of the chromatography conditions can be performed in the manner described for 6x His-tagged proteins and Zn proteins.
Important note
- It is not recommended to use regenerated columns for the initial protein screening. New columns should be used.
- But the regenerated gel can be used for purification of proteins in the optimization step.
6x His-tagged protein purification with PDC using PDC KIT Zinc protein purification with PDC using PDC KIT Ordering information