showForm Immunoprecipitation using IgY

Name:
Phone:
E-mail:
Message

Immunoprecipitation using IgY

Important note: IgY DOES NOT BIND TO neither Protein G nor Protein A, therefore other methods should be considered, as stated below.

  • Method 1:
    Couple IgY antibodies to the matrix/beads.

    Via free amino groups NHS-activated Sepharose (GE Healtcare) or using AffiGel 15 BioRad
    Coupling is done by free amino groups, and the possibility that antibody will be coupled via antigen binding domain must therefore be considered.

    Via sugar groups
    Use CarboLink Pierce or any other suitable product 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 interfere with the antigen binding domain.

    Dynal tosyl-activated magnetic beads (Invitrogen)
    After coupling, 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. This is compare to the use of IgG, where heavy chain is around 50 kDa.

  • Method 2: Using a secondary anti-chicken IgY antibodies from rabbit or goat, which bind to Protein G or Protein A.

________________________________________________________________________________________________

General considerations

  • 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 start with, try a similar concentration to that used in a Western Blot.
  • Isolation of a complex between the antigen of interest and IgY antibodies: IgY antibodies will not bind to neither Protein G nor Protein A. Therefore, use anti-total IgY secondary antibodies (goat, rabbit), conjugated to Protein G (or Protein A)
  • Protein expression: Consider the level of expression of your target protein present in the cell lysate. Is it enough?
  • Amount of the secondary, goat anti-chicken antibody should usually be in 2-5-fold excess over the concentration of chicken antibody, to make 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 destroy the antibody. Extreme pH, as well as the excess of detergent, might also contribute to loss of the antibody activity.

________________________________________________________________________________________________

  • Examples of steps in one of immunoprecipitation protocols using IgY:

    • Step 1: Incubation of a cell supernatant with IgY antibodies. Overnight incubation over at 4°C. In some cases, up to 1.5 M NaCl can be used in this step. Check if your target protein will cope with these conditions. Use of PEG 6000 at 2 % is also recommended to aid immunoprecipitation.
    • Step 2: Adding goat anti-chicken antibody. Incubation 2-4 h on ice
    • Step 3: Adding Protein G Sepharose Incubation by gentle rotation, 30-60 min at 4°C (rocking might be less efficient).

    References:
    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