Caspases,Paracaspases, and Metacaspases Methods and Protocols

169

4 Notes

1. To avoid SDS precipitation, the sample buffer needs to be
   heated prior to use.


2. Initially, concentrated HCl (12 N) can be used to drastically
   reduce the starting pH. As a near to desired pH is reached,
   HCl with lower ionic strengths should be used to avoid a sud-
   den drop below the required pH.


3. Addition of HCl induces an exothermal reaction which in turn
   will affect the pH. Make sure that the fi nal adjustments are
   performed on a room-tempered solution.


4. The solution can be stored for several weeks at 4 °C, or at
   −20 °C.


5. The buffer can be reused up to four times with maintained
   transfer capacity. Store at 4 °C.


6. A 10× transfer buffer stock solution with respect to Tris base
   (248 mM) and glycine (1,918 mM) can be prepared. Dilute
   in 700 mL water and add 200 mL of methanol, store at 4 °C
   until use.


7. The blocking solution does not contain any preservatives and
   the presence of lactic acid may infl uence the pH during pro-
   longed storage. Therefore this buffer should be prepared fresh
   for each blotting procedure. Blocking buffers which contain
   casein (0.5–1 %) that is purifi ed from milk by the Hammarsten
   method can be used as an alternative (Thermo Scientifi c).


8. Most diluted primary antibodies can be reused several times.
   For additional preservation capacity add 0.05 % sodium azide.
   Avoid this buffer when diluting horseradish peroxide (HRP)
   conjugated antibodies as the sodium azide is an inhibitor of
   HRP enzyme activity.


9. The itemized antibodies are targeting human caspases. Only
   caspases which have been described to be involved in apoptosis
   signaling are listed.


10. Heating to approximately 60 °C is required to dissolve para-
    formaldehyde powder in PBS. Once the paraformaldehyde is
    dissolved, the solution should be cooled and fi ltered.
    Formaldehyde is toxic, use only in fume hood.


11. Normal serum from the same species as the secondary anti-
    body (e.g., normal goat serum, normal donkey serum) can be
    used as an alternative to BSA.


12. Only three examples of antibodies targeting common caspase
    substrates are provided here. For a more complete list of cas-
    pase substrates, see ref. [ 8 ] or the CASBAH/MerCASBA, a
    searchable databases of caspase substrates [ 4 , 5 ].

Caspases in Mammalian Cell Cultures