DBI
For research use only
Buffer
Buffer
Membrane bound Protein Extraction Kit
o
(Catalog #DBI-111; DBI-112;Store at 4 C)
I.
II.
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.
Kit Contents:
Component
Buffer A
Buffer B
Protease Inhibitor Cocktail
SDS-PAGE loading buffer(6×)
III.
DBI-111
50
assays
50ml
10ml
1vial
5vial
DBI-112
100
assays
100ml
20ml
1vial
10vial
NM
NM
Gray
green
Membrane Protein Extraction Protocol:
Cell type
Cell Amount
Buffer
Buffer
Before use, aliquot enough Buffer A and Buffer B, 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).
•
The following protocol is described for extraction of ~5-10 x 106
cells. If more cells are used, scale up the volume proportionally.
•
The amount of buffer required for each extraction is dependent
upon the amount of starting cell material.
Other reagent required but not provided : PBS.
Table a: Buffer volumes necessary for extraction of
adherent tissue culture cells
Cell type
Flask/Dish size
DBI
T25
Research
Adherent tissue culture cells
Flask
Dish(mm)
T75
T175
35
60
100
Products
3ml
600ul
0.5ml
50ul
1ml
200ul
2 ml
400ul
A
B
Suspension grown
cells/rozen cell pellet
3 - 5 x 10 6
1 - 2 x 107
cells
cells
1 ml
2 ml
200ul
400ul
Fragmented Tissue*
25 - 50
mg
1 ml
200ul
100 200 mg
2 ml
400ul
For adherent cells, scrape cells in wash buffer and then spin down
(3000 rpm for 5 minutes) to pellet cells.
2.
Resuspend cells in 1ml of the Buffer A in an ice-cold Dounce
homogenizer . Homogenize cells on ice for 30-50 times.
For tissue samples, homogenize tissues in 2-3 volume of the
Buffer A, until it is completed lysed (30-50 times).
Read the entire protocol before beginning the procedure. Be sure
to keep all buffers and reagents on ice at all times during the
experiment.
•
2ml
400ul
B. Extraction of Membrane bound Protein:
1. Collect cells (5-10 x 106) by centrifugation at 600 x g for 5 minutes
o
at 4 C.
A. General Consideration and Reagent Preparation:
•
1ml
200ul
Table b: Buffer volumes necessary for extraction of
suspension grown cells, frozen cell pellets or tissue
Cap code
Cap color
A
B
Note: Efficient homogenization may depend on the cell type. To
check the efficiency of the homogenization, pipette 2-3 μl of the
homogenized suspension onto a cover slip and observe under a
microscope. A shiny ring around the nuclei indicates that cells are
still intact. If 70-80 percent of the nuclei do not have the shiny ring,
proceed to the next step. Otherwise, perform 10-30 additional
passes.
3.
Transfer the homogenate to a microcentrifuge tube. Centrifuge in
o
700 g for 10 minutes at 4 C. Collect supernatant and discard the
pellet.
4.
Transfer the supernatant to a new vial and centrifuge at 10,000g
o
for 30 min at 4 C.
5.
Collect the pellet and discard the supernatant. Resuspend The
o
pellet in 50---200ul Buffer B for 30min at 4 C , centrifuge at
o
10,000g for 30min at 4 C
6. Collect the supernatant (This is Membrane bound Protein
Fraction)
Tel:021-51107991
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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Tel:021-51107991
400-711-6961
www.xinghanbio.com