Hb isolation not requiring protease inhibitor?

[dttom]

My question is just stated on title, in the course of hemoglobin isolation from blood sample, why protease inhibitors don't need to be added? Wouldn't proteases degrade the Hb after cell burst? (I was told that proteases are activated after cell burst, though I don't know the actual mechansim)

[CharonY]

Proteases are not activated by cell lysis, however outside the regulated environment of a cell the proteases can degrade without check everything you got in your vial, plus you can introduce additional ones by contamination. Whether you need an inhibitor depends largely on the extraction method used.

[dttom]

So could you give an example of when inhibitor is not required?

[CharonY]

Under strongly denaturing conditions, for example.

[dttom]

If, procedures concerned just were just conducted at 4deg, should inhibitors be involved? I personally think they should be.

[CharonY]

Again, there is no general rule for that and there is always the question what you want to do and what kind of inhibitors you have in mind. Especially with hemoglobin many chelators (as EDTA) are suboptimal as they damage the metal core of the protein. Low temperature reduce chances of degredation, of course, but then it depends on how fast you work, what buffers you use, etc. There are dozens of different protocols out there, for some applications inhibitors are crucial, for some helpful, other useless and in worst case harmful.

 

Basically it boils down to the question whether inhibitors will hurt downstream. If yes, you have to find an alternative way to do it. If not, adding it may or may not help.