Knowledge
by
experience
by
experience
How to use IgY? General recommendations
IgY present in egg yolk:
- Western Blot - dilute egg yolk 1:1 or 1:2 with PBS pH 7.4.
Centrifuge at room temperature for 30 min. at 4000-6000 x g. Resulting
supernatant can be used directly in dilution series like: 1:100, 1: 1000 and 1: 10 000.
Testing different dilutions is recommended. Western blot membrane should be washed carefully during
whole procedure. Note: Using crude egg yolk in a Western Blot might result in
increased background.
Please, check the protocol:Western Blot using IgY. - ELISA - dilute egg yolk several times with PBS + 0.5 % Tween 20. (1: 10 for example)
Blocking of a plate can be done for 2 hours at room temperature or overnight with 2 % fat free dry milk in PBS + 0.05 % Tween 20 is recommended. Warm up diluted egg yolk to RT before applying on ELISA plate. Incubate egg yolk sample containing primary antibodies for 2 hours at 30ºC or RT. - Immunohistochemistry - crude egg yolk IgY is not recommended to use
directly, since this might result in a rather high background.
AgriSera offers IgY antibodies as egg yolk or purified (total IgY fraction) or affinity purified, as requested.
- Western Blot - test IgY concentration from 0.5 - 15 ug/ml of the antibody
buffer.
Please, check the protocol:Western Blot using IgY. Trouble shooting for Western Blot - ELISA - prepare the dilution series of antibodies using PBS + 0.5 % Tween 20,
proceed as above. Trouble shooting for ELISA
- Immunohistochemistry - from 1 - 20 ug of IgY/ ml of antibody buffer.
Careful optimisation of concentration for each specific antibody is required.
IgY can be used in immunohistochemistry after first purification. If obtained results
are not satisfactory, antibodies require further
affinity purification. However, note that after affinity purification some antibodies, which bind very strongly to the antigen might be lost.
Last changed: 2009-04-02 by WebMaster