Immunoprecipitation using IgY
Important note: IgY DOES NOT BIND TO neither Protein G nor Protein A, therefore other methods should be considered as:
- Method 1:
Couple IgY antibodies to the matrix/beads.
A. via free amino groups NHS-activated Sepharose (GE Healtcare) or using AffiGel 15 BioRad
Coupling is done by free amino groups, therefore a possibility that antibody will be coupled via antigen binding domain must be considered.
B. via sugar groups
Using CarboLink Pierce or any other suitable for this purpose.
IgY is more heavily glycosilated than IgG in the Fc part of the antibody, this allows coupling to be matrix which will not interfeere with antigen binding domain.
C. Dynal tosyl-activated magnetic beads (Invitrogen)
Afterwards follow standard immunoprecipitation protocol used for IgG.
Advantage of this method:
Heavy chain of IgY is ~ 70 [kDa]. This will allow to visualize smaller antigens on Western Blot without masking by heavy chains of IgY immunoglobulin, compare to use of IgG where heavy chain is around 50 [kDa].
- Method 2: Using a secondary anti-chicken IgY antibodies from rabbit or goat which are binding to Protein G or Protein A.
- Optimization of the amount of precipitating antibody (IgY): Amount of IgY antibodies which should be used in the assay needs to be determined empirically. To begin with try similar concentration as the one used in a Western Blot.
- How to isolate a complex between antigen of interest and IgY antibodies? (IgY antibodies will not bind to neither Protein G nor Protein A)
Using anti-total IgY secondary antibodies (goat, rabbit) conjugated to Protein G (or Protein A)
- Consider: Level of expression of your target protein present in cell lysate. Is it enough?
- Note: Amount of the secondary, goat anti-chicken antibody:
Should be usually in 2-5 fold excess over the concentration of chicken antibody, to be sure that all of the chicken antibody-antigen complex is precipitated. 1 ml of the Protein G Sepharose has a capacity to bind 100 femtomoles of goat antibody.
- Avoid: Presence of DTT or other reducing agents in the sample lysate, since they will distroy antibody. Extreme pH as well as the excess of detergent might also contribute to loose of the antibody activity.
Examples of steps in one of immunoprecipitation protocols using IgY:
- First step Incubation of a cell supernatant with IgY antibodies Incubation over night, 4?C In some cases up to 1.5 M NaCl has been used in this step. Check if your target protein will cope with conditions. Use of PEG 6000 at 2 % can be also recommended to aid immunoprecipitation.
- Second step Adding goat anti-chicken antibody Incubation 2-4 h on ice
- Third step Adding Protein G Sepharose Incubation by gentle rotation up 30-60 min. at 4?C (rocking might be less efficient).
References where IgY has been used successfully in immunoprecipitation:
G-J. Lemamy et al. 1999 "High affinity antibodies from hen's-egg yolks against human mannose-6-phosphate/insulin-like growth-factor-II receptor (M6/IGFII-R): characterization and potential use in clinical cancer studies" Int. J. Cancer: 80, 896-902.
G. Camenisch et al. 1999 "General applicability of chicken egg yolk antibodies: the performance of IgY immunoglobulins raised against hypoxia-inducible factor 1 alfa" FASEB J: 13, 81-88