[Protein-analysis] Re: serine protease inhibition by plastic
(by hzhen At freeuk.com)
Tue Nov 21 21:27:20 EST 2006
On 20 Nov 2006 18:40:12 -0800, "rolando"
<rolando.perdomo At infomed.sld.cu> wrote:
>Thanks a lot for your support. I fact I am a beginner using ultrasonic
>for cell disrupting. Now I am very enthusiastic to get more hints about
>the issue. To get an idea: I am studying a serine protease which comes
>from the hemocytes from the lobster (these cells have hardness similar
>to lymphocytes). In the past I used a glass piston homogenizer.
>However, currently I would prefer ultrasonic treatments due to is more
>efficient and less handling would required. I am using a cycle of 30
>sec at 20 Watt for a total of 2 min on ice (300 ul sample). This cycle
>did not seem particularly aggressive to me in the first time. As I am a
>novice ultrasonic user, I tested the cycle using a commercial
>preparation of trypsin, and the activity was not affected by the cycle.
>In addition, other enzyme naturally occurring in the hemocytes, the
>phenoloxidase, was not affected neither. What called me the attention
>was that even compared to glass piston homogenization the serine
>protease activity was lower. That is why I thought that maybe
>extractable materials from plastic could be the cause.
Different proteins have different properties, what happens to one or
more proteins does not mean that another protein will be affected (or
not affected) in similar ways.
> In addition,
>sometime I heard that this could occur. I am considering that maybe
>this serine protease indeed can be less resistance to the heating
>stress caused by the ultrasonic than commercial trypsin and
>phenoloxidase. Perhaps I should reduce the cycles, for instance to 15
>sec, and to work with glass reservoir. I was bought by the brochure of
>the ultrasonic equipment, which emphasize that the microprobe can be
>used with eppy tubes and ultrasonicate very low volume of samples.
15 sec sounds fine to me.
There are of course other methods of lysing cells - glass beads,cell
lysis reagents (a few companies sell them), freeze thaw, osmotic
shock, etc.. Perhaps you can try them and compare to sonication and
see if there is any difference, and that may tell you whether it is
the plastic that is the problem or the sonication.
>Looking forward to your thoughts and comments.
>DK ha escrito:
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