8 replies to this topic

[yobou]

Dear All
a colleague claims that 1% SDS in lysis buffer is sufficient to inhibit proteases and there is no need to use protease inhibitors. is this correct?

[hobglobin]

yobou, on Jul 6 2009, 07:56 PM, said:

Dear All
a colleague claims that 1% SDS in lysis buffer is sufficient to inhibit proteases and there is no need to use protease inhibitors. is this correct?

At least Proteinase K is not inhibited...

[bob1]

It is false - SDS is a detergent, it primarily dissolves lipids rather than proteins.

Edited by bob1, 06 July 2009 - 04:26 PM.

[HomeBrew]

Proteinase K's activity is actually enhanced by SDS: "Activity can be stimulated by addition of denaturing agents (SDS and urea)" (see here, under "Activators").

[yobou]

HomeBrew, on Jul 7 2009, 12:23 PM, said:

Proteinase K's activity is actually enhanced by SDS: "Activity can be stimulated by addition of denaturing agents (SDS and urea)" (see here, under "Activators").

I mean proteases in human cancer cells (serine, cysteine and aaspartate , ...etc proteaes) . I guess proteinaseK is not among them.

[HomeBrew]

Proteinase K is a serine protease from a eukaryotic organism...

Regardless, in answer to your question -- I don't think 1% SDS is effective enough to rely on it alone for protease inhibition.

[GeorgeWolff]

Ask your colleague for the data.

[yobou]

GeorgeWolff, on Jul 8 2009, 06:36 PM, said:

Ask your colleague for the data.

actually before I open this topic for discussion I did so, and he showed me a blot for a protein that is clealry upregulated by certain drug treatment as a positive control. That is why  I  was asking whether this method is valid or not.

Edited by yobou, 08 July 2009 - 12:06 PM.

[Tfal]

is there any reason you wish to avoid habitual protease inhibitors?