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Western Blot using IgY

 

Important note: Please be aware that protocols used for IgG antibodies (rabbit, goat, mice) often must be optimized to get ideal results with IgY antibodies.


1. Blocking unspecific binding

Block unspecific binding by washing the membrane in 25 ml blocking buffer/13x13 cm filer for 2 h at RT 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, but there are complex carbohydrates in milk which will absorb out antibodies that recognize carbohydrate determinants.
  • Some IgY antibodies might recognize milk proteins, which will lead to a 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 background 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.
    Limitation:
    Cannot be used if antibodies were raised to a peptide-BSA conjugate.
  • Serum
    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. Filter wash

Wash each filter briefly, twice, followed by 1 x 15 min and 4 x 5 min at RT in Washing buffer. In case there is still a high background signal, increase the length of the washing steps further. 

  • Washing buffer:
    1 x TBS, 0.05 % Tween-20

3. Addition of primary antibody

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 h at RT (or 37°C, optional).

  • Antibody buffer: 1 x TBS, 2 % milk powder, 0.05 % Tween 20


4. Filter rinse

Rinse the filter briefly twice, followed by 1 x 15 and 4 x 5 min at RT in Washing buffer.


5. Addition of secondary antibody

Add the secondary antibody to the Antibody Buffer. Use, for instance, Agrisera's rabbit anti-IgY,HRP conjugated or goat anti-IgY, HRP conjugated. Start with a dilution of 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. Filter wash

Wash the filter for 4 x 5 min in Washing buffer, followed by 1 x 15 min in dH2O at RT.


Important!

In case of high unspecific background, increase the time of the membrane washes with frequent exchange of the Washing buffer.

Agrisera video tutorial: How to make a good western blot?

In case of high background levels, firstly test if the secondary antibody is 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 nitrocellulose and PVDF in case of problems.


Check also -  Agrisera western blot trouble shooting