DBI
For research use only
Table a: Buffer volumes necessary for extraction of adherent
tissue culture cells
Mammalian Cell(or tissue) Extraction Kit----I type
(Catalog DBI-101; DBI-102; Store at 4℃)
I.
Introduction:
II.
DBI’S Kit provides optimized buffers and reagents for effective
extraction of total celluar proteins from mammalian tissues and cells.
The procedure offers consistent yield and high purity (over 90%). Total
celluar proteins prepared using the kit can be utilized in a variety of
applications, such as Western blotting, 2-D gels, and enzyme analyses,
etc. The entire procedure takes less than 1 hour.
Kit Contents:
Cap
DBI-101 DBI-102
code
Component
Cap
50
100
assays
assays
color
Protein extractor
50ml
100ml
NM
Protease Inhibitor Cocktail(100×)
1vial
1vial
Gray
5vial
10vial
green
SDS-PAGE loading buffer(6×)
III.
Protein Extraction Protocol:
Cell type
Flask/Dish size
T25
Adherent tissue culture cells
Flask
Dish(mm)
T75
T175
35
60
100
Protein extractor
500ul
1ml
3ml
0.3ml
0.5ml
1ml
Protease Inhibitor
Cocktail
5μl
10μl
30μl
3μl
5μl
10μl
Table b: Buffer volumes necessary for extraction of suspension
grown cells, frozen cell pellets or tissue.
Suspension grown cells
frozen cell pellet
Fragmented Tissue
（mg）
Cell Amount
3 - 5 x 10 6
1 - 2 x 107
25 - 50
100 - 200
Protein extractor
500ul
1ml
500ul
1ml
Protease
Inhibitor Cocktail
5 μl
10 μl
5 μl
10 μl
Cell type
A. General Consideration and Reagent Preparation:
•
•
•
•
Read the entire protocol before beginning the procedure. Be sure
to keep all buffers and reagents on ice at all times during the
experiment.
Before use, aliquot enough Protein extractor, add 1/100 volume
of the reconstituted Protease Inhibitor Cocktail （e.g., Add 10μl
to 1ml buffer）to make the buffer Mix. (Note: Some precipitation
may occur after adding the Protease Inhibitor Cocktail. You may
continue using the buffer or simply remove the precipitates by
centrifugation).
B. Protein Extraction Protocol:
The following protocol is described for extraction of ~5 x 106 cells and should
generate ~500ul of cell lysate.
1.
o
Collect cells by centrifugation at 600 x g for 5 minutes at 4 C.
Note: For adherent cells, scrape cells into PBS and spin down to pellet
cells.
2.
wash cell or tissue 2—3 times with PBS.
Resuspend cells in 500μl of the Extraction Buffer Mix. Pipet up and
down several . Note: For tissue samples, homogenize tissues in 1000μl
of the Extraction Buffer Mix, until it is completed lysed.
The following protocol is described for extraction of 5-10×106cells.
If more cells are used, scale up the volume proportionally.
3.
Incubate on ice for 10 minutes, then vertex for 5 seconds.
4.
Centrifuge in a microcentrifuge at 12000rpm for 3 minute.
The amount of buffer required for each extraction is dependent
upon the amount of starting cell material.
Other reagent required but not provided : PBS.
5.
Collect the supernatant (Cell Lysate) and discard the pellet.
6.
Store cell lysate at –70 C for further studies.
DBI
Research
Products
o
Tel:400-711-6961
www.xinghanbio.com
DBI
For research use only
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