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

• 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.
  Limitation:
  Can not be used if antibodies were rised to 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.
    
  
  
  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 rabbit or goat anti-IgY HRP conjugated. Start with the dilution at least 1 :10 000 (even higher dilutions are recommended).
  Cross-reactivity signal coming from a secondary antibody can be easily checked by omitting a primary antibody in the whole procedure.

  6. Wash the filter 4 x 5 minutes in Washing buffer, followed by 1 x 15 minutes in dH2O at room temperature.
  
  Important!
  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 empty transfer membrane, follow the procedure above without the step with primary antibodies. Good results have been obtained using ECL Advance developing reagent for ECL (Amersham Biosciences), since primary antibodies can be used in a very high dilution, often background signal will be diluted out at that step. Also detection of proteins with low endogenous levels can be improved.
  Nitrocellulose membrane will generally give lower background levels.
  Using extra sensitivite development systems can contribute to increased background signals.
  Check also: Western Blot troubleshooting