Clontech Laboratories His60 Ni Superflow Cartridges Kullanıcı Kılavuzu - Sayfa 10

Laboratuvar Ekipmanları Clontech Laboratories His60 Ni Superflow Cartridges için çevrimiçi göz atın veya pdf Kullanıcı Kılavuzu indirin. Clontech Laboratories His60 Ni Superflow Cartridges 15 sayfaları. Purification of expressed his-tagged proteins from bacterial, yeast, mammalian, and baculovirus-infected cells.

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His60 Ni Superflow Cartridges User Manual
V.
Sample Preparation & Purification
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING.
Use this procedure to (a) prepare your his-tagged protein sample for (b) automated or (c) manual purification using His60
Ni Superflow Cartridges.
A.

PROTOCOL: Extracting Proteins from Cells

This procedure has been optimized for extraction of proteins from fresh or frozen cell pellets using xTractor
Buffer. The extraction volumes can be adjusted, as long as
pellet.
1.
2.
3.
PT5143-1
031716
Resuspend the cell pellet
Add
20 ml of xTractor Buffer
fully resuspend the pellet.
Optional step – lysozyme & DNase I/protease inhibitor
Add
40 μl of 1 unit/μl DNase I solution
inhibitor. Mix gently, pipetting up and down several times.
free protease
NOTES:
DNase I reduces the viscosity of the lysate, allowing for more efficient removal of cellular
debris. DNase I can be used without lysozyme. However, if you are treating cells with
lysozyme, you must also treat these cells with DNase I.
Lysozyme helps to fully disrupt bacterial walls and is highly beneficial when extracting high
molecular weight proteins (>40 kDa).
The lysozyme solution may form a precipitate. Resuspend the contents of the bottle and apply
200 μl of suspension directly to the mix or (optionally) centrifuge 200 μl of lysozyme
solution for 5 min at 14,000 rpm & use the supernatant to perform the lysis.
We recommend that you use our ProteoGuard EDTA-Free Protease Inhibitor Cocktail
(Cat. Nos. 635672 & 635673).
Incubation
Incubate with gentle shaking for 10 min at room temperature. (If desired, you may incubate the
solution at 4°C.)
NOTE: At the end of the incubation period, there should be no visible particles. If cell pellet
fragments are present, resuspend them by pipetting the solution up and down and incubating for
an additional 1–2 min.
Clontech Laboratories, Inc. A Takara Bio Company
20 ml of xTractor Buffer
to
pellet. Mix gently. Pipet the mixture up and down to
1 g of cell
and
200 μl of 100X lysozyme
www.clontech.com
are used per
1 g of cell
solution. Add
EDTA-
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