DBI
For research use only
Mammalian Cell Cytosol Protein Extraction Kit
(Catalog DBI-109; DBI-110; Store at 4℃)
I.
II.
III.
Table a: Buffer volumes necessary for extraction of
adherent tissue culture cells
Introduction:
DBI’S Kit provides optimized buffers and reagents for effective
extraction of Membrane bound proteins from mammalian tissues and
cells. The procedure offers consistent yield and high purity (over 90%).
Membrane bound 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-109 DBI-110
code
Component
Cap
50
100
assays
assays
color
10×wash buffer
25ml
100ml
NM
Protein extractor
50ml
100ml
NM
Protease Inhibitor Cocktail(100×)
1vial
1vial
Gray
5vial
10vial
green
SDS-PAGE loading buffer(6×)
Membrane Protein Extraction Protocol:
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).
Cell type
Flask/Dish
size
Protein
extractor
Adherent tissue culture cells
Flask
Dish(mm)
T75
T175
35
60
T25
500ul
1ml
3ml
0.3ml
0.5ml
1ml
5μl
10μl
30μl
3μl
5μl
10μl
Protease
Inhibitor
Cocktail
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
B. Mammalian Protein Extraction Protocol:
6
The following protocol is described for extraction of ~5 x 10 cells and should
generate ~100mg 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 1×wash buffer.
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.
·
Dilute Wash Buffer(10×) to 1×Wash Buffer.
•
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 5seconds.
4.
Centrifuge in a microcentrifuge at 12000rpm for 3minute.
The amount of buffer required for each extraction is dependent
upon the amount of starting cell material.
5.
Collect the supernatant (Cell Lysate) and discard the pellet.
6.
Store cell lysate at –70 C for further studies.
DBI
Research
Products
100
o
Tel:400-711-6961
www.xinghanbio.com
DBI
For research use only
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DBI BIO-TECHNOLOGY RESEARCH CENTER
Telethone: 400-711-6961
Web: www.xinghanbio.com
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