Western Blot using IgY
Important note: Please be aware that protocols used for IgG antibodies (rabbit, goat, mice) often have to be optimised to get best results with IgY antibodies.
1. Block unspecific binding
by washing membrane in 25 ml blocking buffer/13x13 cm filer for 2 hours at room temperature on a shaker. Example of a Blocking buffer (Blotto)
1x TBS (20 mM Tris/HCl, pH 7.5,150 mM NaCl, 3-5 % low-fat dried milk powder, 0.05 % Tween-20 (or Nonidet P-40)
Blotto - Limitations:
- Usually very effective however: there are complex carbohydrates in milk and these will absorb out antibodies that recognize carbohydrate determinants.
- Some IgY antibodies might recognize milk proteins (high background signal).
- 10 % nonfat dry milk might block so efficiently, and in some cases so well, that no bands of interest will be seen.
Problems with backgorund while using nonfat dry milk can be often solved by trying BSA instead.
Blocking insufficient - alternatives to try:
- BSA Partially purified, most antibodies will not cross-react.
Can not be used if antibodies were rised to peptide-BSA conjugate
Some animals from which blocking serum has been obtained may have developed antibodies to the antigen in question. If this is the case, they may bind to the antigen and prevent the primary antibody from binding. Agrisera's blocking serum collection.
Please test different blockers and adjust your protocols accordingly.
2. Wash each filter
briefly, twice, then 1 x 15 minutes and 4 x 5 minutes at RT in Washing buffer. In case of still high background siganl - increase the length of a washing step even more than what is recommended above.
- Washing buffer:
1 x TBS, 0.05 % Tween-20
3. Add primary antibody
to 10-20 ml of Antibody buffer (dilution range from 1:100 to 1: 30 000) and incubate the filter for 1-3 hours at room temperature (or 37°C, optional).
- Antibody buffer: 1 x TBS, 2% milk powder, 0.05 % Tween 20
4. Rinse the filter
briefly twice, following by 1 x 15 and 4 x 5 minutes at RT in Washing buffer.
5. Add secondary antibody
in Antibody Buffer. Use for instance Agrisera's rabbit anti-IgY,HRP conjugated or goat anti-IgY, HRP conjugated. Start with the dilution at least 1 :10 000 (even higher dilutions are recommended). Agrisera secondary antibodies to chicken IgY labelled with: ALP (Alkaline phosphatase), Biotinylated, Fluorescent, HRP (horse radish peroxidase conjugated).
Cross-reactivity signal coming from a secondary antibody can be easily checked by omitting a primary antibody in the whole procedure and developing a membrane.
6. Wash the filter
4 x 5 minutes in Washing buffer, followed by 1 x 15 minutes in dH2O at room temperature.
Amount of membrane washes. In case of high unspecific background, increase the time of the wash with frequent exchange of Washing buffer.
In case of high background levels, firstly test if the secondary antibody is not contributing to the background. Use a small piece of transfer membrane, follow the procedure above without applying primary antibodies. Good results have been obtained using various ECL developing reagents with enhanced chemiluminescence like TMA-6 (Lumigen), WestPico (Pierce). High dilution of primary antibodies can often contribute to reduced background signal, depending upon experimental set up. This way detection of proteins with low endogenous levels can be improved.
Nitrocellulose membrane might give lower background levels, therefore it is worth to test both nitrocelluose and PVDF in case of problems.
Check also - Agrisera western blot trouble shooting