problems with proteases in neutrophil extracts

Dr. Rudolf Oehler rudolf.oehler at akh-wien.ac.at
Mon Sep 7 09:47:32 EST 1998


PROTEASES IN CELL LYSATES OF NEUTROPHILS (PMNs) 
DIGEST ALL MY PROTEIN!

WHAT IS THE IDEAL LYSIS PROTOCOL TO AVOID PROTEASE ACTIVITY???

I have been trying to quantify the expression of a protein in lysates
of PMNs of different patients. In 8 out of 19 extracts i'v got more or
less total degradation of proteins. In the other samples the protein
pattern after SDS-PAGE looked quite good, but bands of proteins with
high molecular weight were not seen in all of these samples. In
western blots I have found partial or total degradation of my protein
in all samples.

Cell lysis protocol used:
PMNs were lysed by hypotonic treatment. The lysis buffer consisted of
10 mM Tris-HCl, pH=7.8, 1 mM EDTA, 10 mM KCl, 0.3% Triton X-100, 1 mM
PMSF. After homogenization, the lysate was collected and centrifuged
at 800 x g for 3 minutes to separate nuclei from the cytoplasm. After
a second centrifugation (17000 x g for 3 minutes) the supernatant,
which contained the cytoplasmic fraction, was stored in aliquots at
-70°C.
This protocol worked well in a number of different cells, but not in
PMNs.

Is there another protocol to try or may be a special one for PMNs???

Thanks in advance.



More information about the Methods mailing list