INSTRUCTIONS
膜蛋白提取试剂盒货号89826
Number Description
89826 Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit, contains sufficient lysis and
extraction reagents for approximately 50-100 mammalian cell pellet fractions containing 5 × 106
cells
each, 15mg of wet yeast cell paste, or 20mg of tissue
Kit Contents:
Mem-PER Reagent A, 10mL
Mem-PER Reagent B, 25mL
Mem-PER Reagent C, 40mL
Storage: Upon receipt store Reagent A at room temperature. Store Reagent B ａnd Reagent C at 4°C.
Product is shipped at ambient temperature.
Note: The ambient shipping conditions may cause Reagent C to appear cloudy upon receipt. Storage at
4°C will clarify the solution. Keep Reagent C at 4°C or on ice at all times.
膜蛋白提取试剂盒货号89826Table of Contents
Introduction ................................................................................................................................................................................. 1
Important Product Information .................................................................................................................................................... 2
Additional Material Required ...................................................................................................................................................... 2
Procedure for Membrane Protein Extraction from Different Sample Types ............................................................................... 2
Protocol 1: Mammalian Cells .................................................................................................................................................. 2
Protocol 2: Yeast Cells ............................................................................................................................................................ 3
Protocol 3: Soft Tissue ............................................................................................................................................................. 3
Protocol 4: Hard Tissue ........................................................................................................................................................... 4
Procedure for Performing a Second Extraction ........................................................................................................................... 4
Preparation of Samples for Downstream Applications ................................................................................................................ 5
A. SDS Polyacrylamide Gel Electrophoresis （PAGE） ........................................................................................................ 5
B. Protein Quantitation ........................................................................................................................................................ 5
C. Other Applications .......................................................................................................................................................... 5
Additional Information ................................................................................................................................................................ 5
Related Thermo Scientific Products ............................................................................................................................................ 6
Introduction
The Thermo Scientific Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit is for the enrichment of integral
membrane proteins from cultured mammalian or yeast cells or from mammalian tissue using a mild detergent-based protocol.
The cells are first lysed with a detergent, after which a second detergent is added to solubilize the membrane proteins. The
cocktail is incubated at 37°C to separate the hydrophobic proteins from the hydrophilic proteins through phase partitioning.
Extraction efficiencies will vary depending on the number of times the integral membrane protein（s） of interest spans the
lipid bilayer. Membrane proteins with up to four transmembrane domains are typically extracted with an efficiency of up to
90%. Cross-contamination of cytosolic proteins into the membrane fraction is typically < 10%.
1312.11
89826
Mem-PER® Eukaryotic Membrane
Protein Extraction Reagent Kit
Pierce Biotechnology PO Box 117 （815） 968-0747 www.thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA （815） 968-7316 fax
2
Important Product Information
• For optimal results, include protease inhibitors （e.g., Product No. 78415） in the Mem-PER Reagents.
• Perform 37°C incubation step in a water bath. Incubator ovens do not heat evenly enough for this application.
• Do not exceed 10 samples in one extraction procedure because rapid resolubilization of the hydrophobic phase into the
hydrophilic phase occurs at room temperature.
• For downstream processing of samples, refer to the Preparation of Samples for Downstream Applications Section
following the main protocols.
Additional Material Required
• Protease inhibitors （e.g., Product No. 78415）
• For soft tissues, a 2mL Dounce Tissue Grinder, such as Kontes or Wheaton Tenbroeck, is required.
• For hard tissues a hand-held homogenizer for 0.5 to 1.5mL samples, such as the Brinkmann Polytron PT-1200CL, is
required.
• Tris Buffered Saline （TBS; 0.025M Tris, 0.15M NaCl; pH 7.2; Product No. 28376） containing protease inhibitors
• Water bath equilibrated to 37°C. Do not use an incubator oven, which will not heat evenly enough for this application.
膜蛋白提取试剂盒货号89826
Procedure for Membrane Protein Extraction from Different Sample Types
Following are four protocols: Protocol 1 is for the extraction of membrane proteins from mammalian cells; Protocol 2 is for
the extraction of membrane proteins from yeast cells: Protocol 3 is for the extraction of membrane proteins from soft tissues;
and Protocol 4 is for the extraction of membrane proteins from hard tissues. （See the Additional Information Section for a
schematic of the mammalian cells ａnd tissue protocols.）
Note: Additional protein recovery may be recovered with a second extraction; see protocol at the end of this section.
Protocol 1: Mammalian Cells
1. Isolate 5 × 106
cells per sample by centrifuging harvested cell suspensions at 850 × g for 2 minutes. Pellet cells （washed
in PBS） in 1.7mL microcentrifuge tubes.
2. Carefully remove ａnd discard the supernatant.
3. Add 150µL of Reagent A to the cell pellet. Pipette up ａnd down to obtain a homogeneous cell suspension. Incubate 10
minutes at room temperature with occasional vortexing.
Note: White, flocculent debris appears upon addition of Reagent A.
Note: To check the cell lysis efficiency, spot 5µL of cell lysate onto a glass slide, add coverslip ａnd view under a light
microscope. Compare with 5µL of the same number of intact cells in 150µL of phosphate-buffered saline （PBS） or Tris-
buffered saline （TBS）.
4. Place lysed cells on ice.
5. Dilute 2 parts Reagent C with 1 part Reagent B, making sufficient mixture for each sample to receive 450µL （e.g., for 10
extractions, combine 3.33mL of Reagent C with 1.67mL of Reagent B）. Keep Reagents B ａnd C at 4°C or on ice at all
times.
6. Add 450µL of diluted Reagent C to each tube of lysed cells ａnd vortex. Incubate tubes on ice for 30 minutes, vortexing
every 5 minutes.
7. Centrifuge tubes at 10,000 × g for 3 minutes at 4°C. Transfer supernatant to new tubes ａnd incubate 10 minutes in 37°C
water bath to separate the membrane protein fraction. To enhance phase separation, increase incubation to 20 minutes.
8. Centrifuge tubes at room temperature for 2 minutes at 10,000 × g to isolate the hydrophobic fraction （i.e., the fraction
containing membrane protein of interest） from the hydrophilic fraction.
Pierce Biotechnology PO Box 117 （815） 968-0747 www.thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA （815） 968-7316 fax
3
9. Carefully remove the hydrophilic phase （top layer） from the hydrophobic protein phase （bottom layer） ａnd save in a new
tube. Perform phase separations as quickly as possible because the interface between the layers slowly disappears at
room temperature.
10. Place the separated fractions on ice. The majority of membrane protein will be in the lower hydrophobic fraction, which
can be used for membrane protein analysis.
Protocol 2: Yeast Cells
1. Prepare approximately 150mg of 405-600µm acid-washed glass beads
2. Harvest yeast cells in the exponential growth phase at a density of 1-5 × 107
cells/mL with an OD600 = 0.3-1.7. Use
approximately, 15mg of wet cell paste per assay.
3. Pellet cells by pulse centrifugation ａnd carefully remove the supernatant.
4. Resuspend cells in 80µL of Reagent A. Pipette up ａnd down to obtain a homogeneous cell suspension.
5. Add the acid-washed glass beads to the cell suspension ａnd vortex for 10 minutes to lyse cells. Pellet the beads by pulse
centrifugation. Transfer the cell suspension into a new microcentrifuge tube ａnd keep on ice.
6. Dilute 2 parts Reagent C with 1 part Reagent B, making sufficient mixture for each sample to receive 720µL （e.g., for 10
extractions, combine 4.8mL of Reagent C with 2.4mL of Reagent B）. Keep Reagents B ａnd C at 4°C or on ice at all
times.
7. Add 720µL of diluted Reagent C into the tube containing the glass beads ａnd briefly vortex to wash. Perform a pulse
spin to gather beads.
8. Transfer wash into tube containing the cell suspension ａnd incubate on ice for 30 minutes, vortexing every five minutes.
9. Centrifuge at 10,000 × g for three minutes at 4°C. Transfer supernatant to new tubes ａnd incubate 10 minutes in 37°C
water bath to separate the membrane protein fraction. To enhance phase separation, increase incubation to 20 minutes.
10. Centrifuge tubes at room temperature （RT） for two minutes at 10,000 × g to isolate the hydrophobic fraction （i.e., the
fraction containing the membrane protein of interest） from the hydrophilic fraction.
11. Carefully remove hydrophilic phase （top layer） from the hydrophobic phase （bottom layer） ａnd save in a new tube.
Perform phase separations as quickly as possible because the interface between layers slowly disappears at RT.
12. Place the separated fractions on ice. The majority of membrane proteins will be in the lower hydrophobic fraction, which
can be used for membrane protein analysis.
Protocol 3: Soft Tissue
1. Place 20mg of soft tissue in a 1.5mL microcentrifuge tube. Add 200µL of TBS to tissue, vortex briefly ａnd discard wash.
2. Transfer rinsed tissue to a 2mL tissue grinder. Add 200µL of TBS to the tissue ａnd homogenize until an even suspension
is obtained （~6 to 10 strokes）.
3. Transfer homogenate to a new 1.5mL tube ａnd centrifuge at 1000 × g for 5 minutes at 4°C.
4. Discard supernatant ａnd resuspend the pellet in 150µL of Reagent A. Pipette up ａnd down to obtain a homogeneous
suspension. Incubate 10 minutes at room temperature with occasional vortexing.
Note: White, flocculent debris appears upon addition of Reagent A.
5. Place lysed cells on ice.
6. Dilute 2 parts Reagent C with 1 part Reagent B, making sufficient mixture for each sample to receive 450µL （e.g., for 10
extractions, combine 3.33mL of Reagent C with 1.67mL of Reagent B）. Keep Reagents B ａnd C at 4°C or on ice at all
times.
7. Add 450µL of diluted Reagent C to each tube of lysed cells ａnd vortex. Incubate tubes on ice for 30 minutes, vortexing
every 5 minutes.
8. Centrifuge tubes at 10,000 × g for 3 minutes at 4°C. Transfer supernatant to new tubes ａnd incubate 10 minutes in 37°C
water bath to separate the membrane protein fraction. To enhance phase separation, increase incubation to 20 minutes.
Pierce Biotechnology PO Box 117 （815） 968-0747 www.thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA （815） 968-7316 fax
4
9. Centrifuge tubes at room temperature （RT） for 2 minutes at 10,000 × g to isolate the hydrophobic fraction （i.e., the
fraction containing membrane protein of interest） from the hydrophilic fraction.
10. Carefully remove hydrophilic phase （top layer） from the hydrophobic protein phase （bottom layer） ａnd save in a new
tube. Perform phase separations as quickly as possible because the interface between layers slowly disappears at RT.
11. Place the separated fractions on ice. The majority of membrane protein will be in the lower hydrophobic fraction, which
can be used for membrane protein analysis.
Protocol 4: Hard Tissue
1. Place 20mg of hard tissue in a 2.0mL microcentrifuge tube. Add 500µL of TBS to the tissue, vortex briefly ａnd discard
the wash.
2. Add 500µL of TBS to the tissue ａnd cut the tissue into small pieces with a clean razor blade.
3. Homogenize minced tissue with a hand-held Polytron®, using a low setting to prevent foaming. Transfer homogenate to
a new 1.5mL tube ａnd centrifuge at 1000 × g for 5 minutes at 4°C.
4. Discard the supernatant ａnd resuspend the pellet in 150µL Reagent A. Pipette up ａnd down to obtain a homogeneous
suspension. Incubate 10 minutes at room temperature with occasional vortexing.
Note: White, flocculent debris appears upon addition of Reagent A.
5. Place lysed cells on ice.
6. Dilute 2 parts Reagent C with 1 part Reagent B, making sufficient mixture for each sample to receive 450µL （e.g., for 10
extractions, combine 3.33mL of Reagent C with 1.67mL of Reagent B）. Keep Reagents B ａnd C at 4°C or on ice at all
times.
7. Add 450µL of diluted Reagent C to each tube of lysed cells ａnd vortex. Incubate tubes on ice for 30 minutes, vortexing
every 5 minutes.
8. Centrifuge tubes at 10,000 × g for 3 minutes at 4°C. Transfer supernatant to new tubes ａnd incubate 10 minutes in 37°C
water bath to separate the membrane protein fraction. To enhance phase separation, increase incubation to 20 minutes.
9. Centrifuge tubes at room temperature for 2 minutes at 10,000 × g to isolate the hydrophobic fraction （i.e., the fraction
containing membrane protein of interest） from the hydrophilic fraction.
10. Carefully remove the hydrophilic phase （top layer） from the hydrophobic protein phase （bottom layer） ａnd save in a new
tube. Perform the phase separations as quickly as possible because the interface between the layers slowly disappears at
room temperature.
11. Place the separated fractions on ice. The majority of membrane protein will be in the lower hydrophobic fraction, which
can be used for membrane protein（s） analysis.
Procedure for Performing a Second Extraction
Although typically not necessary, a second extraction may result in additional protein recovery.
1. Determine volume of the hydrophilic fraction ａnd add an equal volume of undiluted Reagent C to this fraction and
vortex.
2. Incubate tubes on ice for 30 minutes, vortexing every 5 minutes during the incubation.
3. Incubate tubes for 10 minutes in a 37°C water bath to separate the membrane protein fraction. To enhance phase
separation, increase incubation to 20 minutes.
4. Centrifuge tubes at room temperature for 2 minutes at 10,000 × g to isolate the hydrophobic fraction from the
hydrophilic fraction.
5. Carefully remove the hydrophilic phase （top layer） from the hydrophobic protein phase （bottom layer） ａnd save in a new
tube. Perform phase separations as quickly as possible because the interface between layers slowly disappears at room
temperature. Place separated fractions on ice.
6. Combine hydrophobic fraction from the first extraction with the hydrophobic fraction from the second extraction.
Pierce Biotechnology PO Box 117 （815） 968-0747 www.thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA （815） 968-7316 fax
5
Preparation of Samples for Downstream Applications
A. SDS Polyacrylamide Gel Electrophoresis （PAGE）
To determine extraction efficiency, perform SDS-PAGE ａnd identify the protein（s） of interest by Western blotting. Dilute
membrane fractions 2- to 5-fold to prevent band ａnd lane distortion caused by high concentrations of detergent. Use Reagent
B diluted 4-fold with ultrapure water for dilution and/or normalization. Alternatively, use the Thermo Scientific Pierce SDS-
PAGE Sample Prep Kit （Product No. 89888） to decrease detergent in the membrane fraction. Use this kit if the protein of
interest is in low abundance ａnd a large volume is required for adequate detection; for samples prepared using two
extractions; or for proteins that migrate at or near the dye front because migrating detergent at the same location causes
severe band distortion.
B. Protein Quantitation
To directly quantify proteins in the hydrophilic fraction, use a protein assay, such as the Pierce
® BCA Protein Assay
（Product No. 23225）, Micro BCA™ Protein Assay （Product No. 23235）, Coomassie Plus （Bradford） Protein Assay （Product
No. 23236） or Pierce 660nm Protein Assay （Product No. 22660）.
To quantify proteins in the hydrophobic fraction, first decrease detergent in the membrane fraction to prevent interference. If
performing subsequent SDS-PAGE, we recommend using the Pierce SDS-PAGE Sample Prep Kit （Product No. 89888）
followed directly by the Pierce BCA Protein Assay Kit. If performing applications other than SDS-PAGE after quantitation,
see Section C below.
C. Other Applications
If the hydrophobic fraction requires decreased detergent for other applications, such as functional studies,
immunoprecipitation or quantitation, an alternative option is to use Pierce Detergent Removal Spin Columns （Product No.
87776-7）. The sample:resin ratio will need to be determined empirically: If too little detergent is removed, the remaining
detergent may interfere with the downstream application. If too much detergent is removed, protein precipitation may occur.
Additional Information
Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit is for the enrichment of integral membrane proteins using a
mild detergent-based protocol. For mammalian cells, a detergent is used to first lyse cells, ａnd then a second detergent is
added to solubilize the membrane proteins. The cocktail is incubated at 37°C to separate the hydrophobic proteins from the
hydrophilic proteins through phase partitioning （Figure 1）. Yeast cells require the use of acid-washed glass beads for cell
disruption. Hard ａnd soft tissues also require mechanical means for disruption （Figure 2）.
Figure 1. Summary of membrane protein extraction from mammalian cells.
Pierce Biotechnology PO Box 117 （815） 968-0747 www.thermoscientific.com/pierce
3747 N. Meridian Road Rockford, lL 61105 USA （815） 968-7316 fax
6
Figure 2. Summary of membrane protein extraction from soft ａnd hard tissues.
Related Thermo Scientific Products
87785 Halt Protease Inhibitor Cocktail, EDTA-Free （100X）, 1mL
87786 Halt Protease Inhibitor Cocktail （100X）, 1mL
78441 Halt Protease ａnd Phosphatase Inhibitor Cocktail, EDTA-Free （100X）, 1mL
78440 Halt Protease ａnd Phosphatase Inhibitor Cocktail （100X）, 1mL
78420 Halt Phosphatase Inhibitor Cocktail （100X）, 1mL
23225 Pierce BCA Protein Assay Kit
89888 Pierce SDS-PAGE Sample Prep Kit
87780 Pierce Detergent Removal Resin, 10mL
78840 Subcellular Protein Fractionation Kit
34095 SuperSignal
® West Femto Maximum Sensitivity Substrate, 100mL
34075 SuperSignal West Dura Extended Duration Chemiluminescent Substrate, 100mL
34080 SuperSignal West Pico Chemiluminescent Substrate, 500mL
Polytron is a trademark of Kinematica AG Company.
This product （“Product”） is warranted to operate or perform substantially in conformance with published Product specifications in effect at the time of sale,
as set forth in the Product documentation, specifications and/or accompanying package inserts （“Documentation”） ａnd to be free from defects in material and
workmanship. Unless otherwise expressly authorized in writing, Products are supplied for research use only. No claim of suitability for use in applications
regulated by FDA is made. The warranty provided herein is valid only when used by properly trained individuals. Unless otherwise stated in the
Documentation, this warranty is limited to one year from date of shipment when the Product is subjected to normal, proper ａnd intended usage. This
warranty does not extend to anyone other than the original purchaser of the Product （“Buyer”）.
No other warranties, express or implied, are granted, including without limitation, implied warranties of merchantability, fitness for any particular
purpose, or non infringement. Buyer’s exclusive remedy for non-conforming Products during the warranty period is limited to replacement of or
refund for the non-conforming Product（s）.
There is no obligation to replace Products as the result of （i） accident, disaster or event of force majeure, （ii） misuse, fault or negligence of or by Buyer, （iii）
use of the Products in a manner for which they were not designed, or （iv） improper storage ａnd handling of the Products.
Current product instructions are available at www.thermoscientific.com/pierce. For a faxed copy, call 800-874-3723 or contact your local distributor.
© 2011 Thermo Fisher Scientific Inc. All rights reserved. Unless otherwise indicated, all trademarks are property of Thermo Fisher Scientific Inc. ａnd its
subsidiaries. Printed in the USA.
