2024年11月2日发(作者：仪夏烟)
ab65400
Plasma Membrane
Protein Extraction Kit
Instructions for Use
For the rapid and sensitive extraction and
purification of Plasma Membrane proteins from
cultured cells and tissue samples.
This product is for research use only and is not
intended for diagnostic use.
Version: 5 Last Updated: 11 April 2014
1
Table of Contents
ew
ol Summary
ents and Storage
Protocol
3
4
5
7
2
ew
The Membrane Protein Extraction Kit provides optimized buffers and
reagents for effective extraction of membrane proteins from
mammalian tissues and cells.
Unlike other available procedures that can only extract the total
cellular membrane proteins (combinations of plasma and organelle
membrane proteins), Abcam’s kit was designed to not only extract
the total cellular membrane proteins, but also purify the plasma
membrane proteins specifically.
The procedure offers consistent yield and high purity (over 90%).
Membrane 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.
3
ol Summary
Sample Preparation
Extraction of Total Cellular Membrane Proteins
Purification of Plasma Membrane Proteins (Optional)
4
ents and Storage
Components
ItemQuantity
Homogenize Buffer
Upper Phase Solution
Lower Phase Solution
Protease Inhibitor Cocktail (Lyophilized)
100 mL
20 mL
20 mL
1 vial
* Store kit at -20°C. Read the entire protocol before beginning the
procedure. Be sure to keep all buffers and reagents on ice at all
times during the experiment.
PROTEASE INHIBITOR COCKTAIL: Reconstitute Protease Inhibitor
Cocktail by adding 250 μl of DMSO, mix well.
HOMOGENIZE BUFFER MIX: Aliquot enough Homogenize Buffer,
add 1/500 volume of the reconstituted Protease Inhibitor Cocktail
(e.g., Add 10 μl to 5 ml buffer) to make the Homogenize 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.
5
onal Materials Required
Microcentrifuge
Pipettes and pipette tips
Dounce homogeniser
PBS
Triton-X-100
6
Protocol
The following protocol is described for extraction of ~5-10 x 10
8
cells.
If more cells are used, scale up the volume proportionally.
tion of Total Cellular Membrane Proteins
t cells ~1 g wet weight (0.2-10 x 10
8
) by centrifugation
at 600 x g for 5 minutes at +4°C.
For adherent cells, scrape cells in PBS and then spin down
(3000 rpm for 5 minutes) to pellet cells.
cells once with 3 ml of ice cold PBS.
-suspend cells in 2 ml of the Homogenize Buffer Mix in an
ice-cold Dounce homogenizer. Homogenize cells on ice for
30-50 times.
For tissue samples, homogenize tissues in 2 volume of the
1X Homogenize Buffer, until it is completed lysed (30-50 times).
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.
7
er the homogenate to a 1.5 ml microcentrifuge tube.
Centrifuge in 700 x g for 10 minutes at +4°C. Collect supernatant
and discard the pellet.
er the supernatant to a new vial and centrifuge at
10,000 x g for 30 min at +4°C.
t supernatant (This is the Cytosol Fraction). The pellet is
the total cellular membrane protein (containing proteins from
both plasma membrane and cellular organelle membrane).
Note:
You may stop here if you only need the total cellular membrane
proteins. If you would like to further isolate the plasma membrane
proteins specifically, continue to section B.
cation of Plasma Membrane Proteins
-suspend the total membrane proteins pellet in 200 μl of the
Upper Phase Solution. Add 200 μl of the Lower Phase Solution.
Mix well and incubate on ice for 5 minutes (Mark the tube as A).
e a fresh phase tube without samples. Adding 200 μl of
Upper Phase Solution and 200 μl of Lower Phase Solution (Mark
the tube as B).
fuge both A & B tubes in a microcentrifuge at 3500 rpm
(1000 x g) for 5 minutes.
8
lly transfer the upper phase from tube A to a new tube
(tube C), keep on ice.
maximize the yield, extract the tube A lower phase again by
adding 100 μl of the Upper Phase Solution from tube B. Mix well
and centrifuge at 3500 rpm (1000 x g) for 5 minutes.
lly collect the upper phase. Combine with the upper phase
from Step 4 (tube C). Extract the combined upper phase by
adding 100 μl of the Lower Phase Solution from tube B, Mix well
and centrifuge at 3500 rpm (1000 x g) for 5 minutes.
lly collect the upper phase. Dilute the upper phase in
5 volume of water. Keep on ice for 5 minutes.
at top speed at a microcentrifuge tube for 10 minutes at
+4°C. Remove the supernatant. The pellet is the plasma
membrane protein.
the plasma membrane proteins at -80°C for further studies.
The membrane fraction can be dissolved in 0.5% Triton X-100 in
PBS or other buffers before use. Generally 30-100 μg plasma
membrane proteins can be obtained.
9
10
UK, EU and ROW
Email: technical@
Tel: +44 (0)1223 696000
US, Canada and Latin America
Email: cal@
Tel: 888-77-ABCAM (22226)
China and Asia Pacific
Email: cal@
Tel: 1 (中國聯通)
Japan
Email: technical@
Tel: +81-(0)3-6231-0940
11
Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.
2024年11月2日发(作者：仪夏烟)
ab65400
Plasma Membrane
Protein Extraction Kit
Instructions for Use
For the rapid and sensitive extraction and
purification of Plasma Membrane proteins from
cultured cells and tissue samples.
This product is for research use only and is not
intended for diagnostic use.
Version: 5 Last Updated: 11 April 2014
1
Table of Contents
ew
ol Summary
ents and Storage
Protocol
3
4
5
7
2
ew
The Membrane Protein Extraction Kit provides optimized buffers and
reagents for effective extraction of membrane proteins from
mammalian tissues and cells.
Unlike other available procedures that can only extract the total
cellular membrane proteins (combinations of plasma and organelle
membrane proteins), Abcam’s kit was designed to not only extract
the total cellular membrane proteins, but also purify the plasma
membrane proteins specifically.
The procedure offers consistent yield and high purity (over 90%).
Membrane 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.
3
ol Summary
Sample Preparation
Extraction of Total Cellular Membrane Proteins
Purification of Plasma Membrane Proteins (Optional)
4
ents and Storage
Components
ItemQuantity
Homogenize Buffer
Upper Phase Solution
Lower Phase Solution
Protease Inhibitor Cocktail (Lyophilized)
100 mL
20 mL
20 mL
1 vial
* Store kit at -20°C. Read the entire protocol before beginning the
procedure. Be sure to keep all buffers and reagents on ice at all
times during the experiment.
PROTEASE INHIBITOR COCKTAIL: Reconstitute Protease Inhibitor
Cocktail by adding 250 μl of DMSO, mix well.
HOMOGENIZE BUFFER MIX: Aliquot enough Homogenize Buffer,
add 1/500 volume of the reconstituted Protease Inhibitor Cocktail
(e.g., Add 10 μl to 5 ml buffer) to make the Homogenize 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.
5
onal Materials Required
Microcentrifuge
Pipettes and pipette tips
Dounce homogeniser
PBS
Triton-X-100
6
Protocol
The following protocol is described for extraction of ~5-10 x 10
8
cells.
If more cells are used, scale up the volume proportionally.
tion of Total Cellular Membrane Proteins
t cells ~1 g wet weight (0.2-10 x 10
8
) by centrifugation
at 600 x g for 5 minutes at +4°C.
For adherent cells, scrape cells in PBS and then spin down
(3000 rpm for 5 minutes) to pellet cells.
cells once with 3 ml of ice cold PBS.
-suspend cells in 2 ml of the Homogenize Buffer Mix in an
ice-cold Dounce homogenizer. Homogenize cells on ice for
30-50 times.
For tissue samples, homogenize tissues in 2 volume of the
1X Homogenize Buffer, until it is completed lysed (30-50 times).
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.
7
er the homogenate to a 1.5 ml microcentrifuge tube.
Centrifuge in 700 x g for 10 minutes at +4°C. Collect supernatant
and discard the pellet.
er the supernatant to a new vial and centrifuge at
10,000 x g for 30 min at +4°C.
t supernatant (This is the Cytosol Fraction). The pellet is
the total cellular membrane protein (containing proteins from
both plasma membrane and cellular organelle membrane).
Note:
You may stop here if you only need the total cellular membrane
proteins. If you would like to further isolate the plasma membrane
proteins specifically, continue to section B.
cation of Plasma Membrane Proteins
-suspend the total membrane proteins pellet in 200 μl of the
Upper Phase Solution. Add 200 μl of the Lower Phase Solution.
Mix well and incubate on ice for 5 minutes (Mark the tube as A).
e a fresh phase tube without samples. Adding 200 μl of
Upper Phase Solution and 200 μl of Lower Phase Solution (Mark
the tube as B).
fuge both A & B tubes in a microcentrifuge at 3500 rpm
(1000 x g) for 5 minutes.
8
lly transfer the upper phase from tube A to a new tube
(tube C), keep on ice.
maximize the yield, extract the tube A lower phase again by
adding 100 μl of the Upper Phase Solution from tube B. Mix well
and centrifuge at 3500 rpm (1000 x g) for 5 minutes.
lly collect the upper phase. Combine with the upper phase
from Step 4 (tube C). Extract the combined upper phase by
adding 100 μl of the Lower Phase Solution from tube B, Mix well
and centrifuge at 3500 rpm (1000 x g) for 5 minutes.
lly collect the upper phase. Dilute the upper phase in
5 volume of water. Keep on ice for 5 minutes.
at top speed at a microcentrifuge tube for 10 minutes at
+4°C. Remove the supernatant. The pellet is the plasma
membrane protein.
the plasma membrane proteins at -80°C for further studies.
The membrane fraction can be dissolved in 0.5% Triton X-100 in
PBS or other buffers before use. Generally 30-100 μg plasma
membrane proteins can be obtained.
9
10
UK, EU and ROW
Email: technical@
Tel: +44 (0)1223 696000
US, Canada and Latin America
Email: cal@
Tel: 888-77-ABCAM (22226)
China and Asia Pacific
Email: cal@
Tel: 1 (中國聯通)
Japan
Email: technical@
Tel: +81-(0)3-6231-0940
11
Copyright © 2012 Abcam, All Rights Reserved. The Abcam logo is a registered trademark.
All information / detail is correct at time of going to print.