2514 Notes
- P1 refers to the residue situated N-terminus to the scissile
bond cleaved by the protease extending towards the
N-terminus (positions P2, P3, etc.) of the peptide, whereas
prime positions (P1′, P2′, etc.) are situated C-terminus to the
cleaved bond [ 22 ]. - We have only used the TNT ® Rabbit Reticulocyte Lysate so
far, but the TNT ® Wheat Germ Lysate should be equivalent.
We routinely use the pDEST (Gateway ® , Invitrogen) vectors
and pUNI51 vectors (SSP consortium). Any vector with the
minimal requirements of a T3 or T7 promoter and terminator
should in theory be suitable. - At this concentration DTT effectively prevents oxidation of
metacaspase cysteins and also prevents formation of intra- and
intermolecular disulfi de bonds, thus inhibiting aggregation of
the protein. Metacaspase structure can be additionally stabi-
lized in the presence of 50 mM CaCl 2 , but it might also lead
to increased autoprocessing. - To prevent aggregation of the protein it is preferable to per-
form dialysis at 4 °C gradually decreasing the concentration of
urea in the dialysis buffer and increasing its volume. - Metacaspases are highly active proteases, thus already 3 ng of
purifi ed metacaspase is enough to produce detectable amount of
cleaved fl uorescent product in activity assay using AMC- tagged
substrates. Still the exact amount of metacaspase required for
the assay might vary depending on purity of the preparation. - Even minute contamination with calcium will signifi cantly
infl uence results of the experiment. An inactive mutant meta-
caspase with substitution of Cys and/or His in the active cen-
ter to Ala or Gly can be used as an alternative negative control
in this assay. It is also known that autoprocessing of metacas-
pases is essential for their proteolytic activity; thus uncleavable
mutant of metacaspase can be used as another negative control
in the activity assay. - This stripping method works for PVDF membranes and usu-
ally allows to effi ciently strip only secondary antibodies. It is
therefore preferable to use for second round of staining pri-
mary antibody produced in a different organism than antibody
used at the fi rst staining. - Avoid uneven destaining, which might impact loading quanti-
fi cation. If necessary the membrane can be re-stained. Let it
dry in vertical position. - Depending on tissue or treatment, expression pattern in the
sample can vary signifi cantly. Take this into consideration
while selecting bands for loading quantifi cation to compare
between samples.
Plant Metacaspases