problems with proteases in neutrophil extracts

M. Johan Broekman, PhD mjbroek at mail.med.cornell.edu
Tue Sep 8 05:59:11 EST 1998


We have used pretreatment of PMN with DFP (diisopropylfluorophosphate).
It's nasty stuff, and goes bad relatively soon, but worked for us.
Problem with PMSF is that it inactivates proteases only very slowly.

Dr. Rudolf Oehler wrote:

> 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.



--
____________________________________
M. Johan Broekman
Cornell Univ Med College
New York, NY
email mjbroek at mail.Please.use.newsgroup.med.cornell.edu





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