Performing a Separation with HiTrap IgY Purifcation HP

Extracted from Affinity Chromatography Principles and Methods, GE Healthcare, 2007

Purifcation Option

  Binding capacity Maximum operating flow Comments
HiTrap IgY Purification HP 100 mg pure IgY/column (1/4 egg yolk) 20 ml/min Purification of IgY from egg yolk. Prepacked 5 ml column.

HiTrap IgY Purification HP columns are packed with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose High Performance). The interaction between the protein and the ligand has been suggested to result from the combined electron donating- and accepting-action of the ligand in a mixed mode hydrophobic-hydrophilic interaction.

Purification Example

Figure 20 shows the purification of a-Hb IgY from 45 ml of egg yolk extract (corresponding to one quarter of a yolk) and Figure 21 shows the SDS-PAGE analysis indicating a purity level of over 70%.

Purifcation of avian IgY on HiTrap IgY Purifcation HP

Fig. 20. Purifcation of avian IgY on HiTrap IgY Purifcation HP.

SDS-PAGE of non-reduced samples on PhastSystem, using PhastGel 4–15%, Coomassie™ staining

Fig. 21. SDS-PAGE of non-reduced samples on PhastSystem, using PhastGel 4–15%, Coomassie™ staining.

Performing a Separation

Column: HiTrap IgY Purifcation HP

Recommended flow rate: 5 ml/min

Binding buffer: 20 mM sodium phosphate, 0.5 M K2SO4, pH 7.5

Elution buffer: 20 mM sodium phosphate, pH 7.5

Wash buffer: 20 mM sodium phosphate, pH 7.5 with 30% isopropanol

As much as possible of the egg yolk lipid must be removed before purification. Water or polyethylene glycol can be used to precipitate the lipids. Precipitation with water is described below.

Precipitation of the egg yolk lipid using water

  1. Separate the egg yolk from the egg white.
  2. Add nine parts of distilled water to one part egg yolk.
  3. Mix and stir slowly for 6 hours at +4 °C.
  4. Centrifuge at 10 000 g, at +4 °C for 25 minutes to precipitate the lipids.
  5. Collect the supernatant containing the IgY.
  6. Slowly add K2SO4 to the sample, stirring constantly, to reach a concentration of 0.5 M.
  7. Adjust pH to 7.5.
  8. Pass the sample through a 0.45 µm filter immediately before applying it to the column.

Purification

  1. Wash the column with at least 5 column volumes of binding, elution and wash buffer.
  2. Equilibrate with 5 column volumes of binding buffer.
  3. Apply the sample.
  4. Wash with at least 10 column volumes of binding buffer or until no material appears in the eluent, as monitored at A280.
  5. Elute the IgY with 10 column volumes of elution buffer.
  6. Wash the column with 8 column volumes of wash buffer.
  7. Immediately re-equilibrate the column with 5 column volumes of binding buffer.

To improve recovery of total IgY or a specific IgY antibody, replace 0.5 M K2SO4 with 0.6–0.8 M Na2SO4. The sample should have the same concentration of Na2SO4 as the binding buffer.

An increase in salt concentration will reduce the purity of the eluted IgY.

The purity of the eluted IgY may be improved by using gradient elution with, for example, a linear gradient 0–100% elution buffer over 10 column volumes, followed by elution with 100% elution buffer for a few column volumes.

To increase capacity, connect several HiTrap IgY Purification HP columns in series. A HiTrap column can be used with a syringe, a peristaltic pump or connected to a liquid chromatography system such as ÄKTAprime plus.

Reuse of HiTrap IgY Purification HP depends on the nature of the sample. To prevent cross-contamination, columns should only be reused with identical samples.

Media Characteristics

  Ligand and density pH stability* Mean particle size
HiTrap IgY Purification HP 2-mercaptopyridine 3 mg/ml Long term 3–11 Short term 2–13 34 µm

* Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

Storage

Wash the column with 5 column volumes 20% ethanol and store at +4 to +8 °C.

Materials

     

Related Links