작성일 : 19-10-08
How to Choose Membrane Protein Isolation Kits
 글쓴이 : 운영자
 

 

How to Choose Membrane Protein Isolation Kits

 

Plasma membrane(PM) proteins play a critical role in a variety of physiological and pathological processes.

Signal transduction, molecular transport and cell-cell interactions are all mediated by PM proteins. PM

proteins include a variety of important proteins such as neurotransmitter receptors, G-proteins, carriers,

voltage-gated ion channels, CD antigens and many drug targets. The detection, characterization, and

intracellular trafficking of PM proteins are, therefore, essential forunderstanding of biological systems.

 

Isolation/purification is usually the first step for characterization and profiling of PM proteins.  However, it has

been proven to be particularly challenging because of their low abundance and the nature of inter-

connectivity of intracellular membrane systems. PM proteins are traditionally isolated by sucrose density

ultra-centrifugation [1,2].  The protocol is tedious and time consuming and takes hours to even days to

complete.  In recent years, more and more publicationscited commercial kits for PM protein isolation and

characterization due to their ease of use and speed.  However, different kits employee different mechanisms

of action.  The efficacy of the kit varies significantly depending upon specific downstream applications.  Due

to the availability of multiple PM protein isolation/purification kits in the market, it is, sometimes, difficult or

even confusing for selection of a proper kit for a particular research project.  In this context, we attemptto

summarize pro and cons of some of the commonly used membrane protein isolation kits and provide a

general guide for selection of commercial membrane protein isolation kits.

 

Generally speaking, all commercially available membrane protein isolation kits can be classified into four

basic categories according to their mechanisms of action or principles of isolation.

1. Phase extraction.  Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo Fisher), Mem-PER®

   Eukaryotic Membrane Protein Extraction Reagent Kit (Thermo Fisher) and ProteoExtract™ Native

   Membrane Protein Extraction Kit (Millipore-Sigma) are typical kits included in this class.  Cells/tissues are

   first lysed by lysis buffer, soluble proteins and insoluble fractions are separated by centrifugation.

   Membrane proteins are further extracted from insoluble fraction by an extractionbuffer based on

   hydrophobicity of membrane proteins.  These kits are relative simple and rapid (about 1h).  The protocol

   generates two distinctive portions: cytosolic fraction and membrane protein fraction however it is not clear

   from the protocol whether a detergent is used for cell lysis. Membrane proteins extracted are derived from

   all membrane systems such as mitochondria, ER, Golgi and nuclei.  What extracted by these kits are

   actually total membrane proteins. It can be anticipated that themembrane protein extraction may or may not

   be complete for certain samples especially for those proteins that transverse bio-membrane multiple

   times. It is not clear if membrane associated proteins remain bound and intact in the extracted membrane

   proteins.

2. Cell surface labeling. Pierce cell surface protein isolation kit (Thermo Fisher) and Qproteomic Plasma

   membrane kit (Qiagen) belong to this class.  A typical experiment using pierce cell surface protein

   isolation kit involves labeling of cell surface proteins by sulfo-NHS-biotin.  After labeling, cells are lysed

   and the cell lysate is applied to an avidin-conjugated solid phase.  Biotinylated plasma membrane proteins

   are eluted using a denaturing elution buffer.  In theory,this approach should produce highly purified plasma

   membrane proteins. However, in reality, This method suffers many inherited disadvantages.  For example,

   not every protein on the cell surface will be labeled.  The protein profile of a given cell culture may change

   with cultured time and conditions.  Steric hindrance and lack of primary amines may interfere with protein

   labeling resulting in inconsistent results. The use of denatured elution solution also limits the application of

   isolated proteins.

3. Phase partitioning. A typical example of this class is the Plasma Membrane Protein Extraction Kit from

   Biovision and Abcam. The mechanisms of action is similar to an earlier published aqueous two phase

   partitioning method [3].  The protocol takes the advantage of differential partitioning of plasma membrane in

   the upper phase and other membranes (such as ER, Golgi and mitochondria) in lower phase for

   enrichment and isolation of plasma membrane proteins. Cultured cells/tissue samplesare first

   homogenized using a Dounce homogenizer.  The lysed samples are subject to multiple extraction and

   centrifugations resulting in total membrane protein, cytosolic and plasma membrane protein fractions.  The

   pros of the method are to be able to isolate detergent-free plasma membrane proteins and the protocol is

   relatively sample and rapid (about 1 -1.5 h).  However, large cell numbers are required (50-100

   millions/sample) and the yield is relatively low (1-100ug/sample).  It was claimedby the manufacturer that

   the purity of plasma membrane protein is over 90% but no supporting data are shown.

4. Spin column-based subcellular fractionation.  This is a next generation plasma membrane protein

   isolation technology from Invent biotechnologies featuring a simple and rapid method for subcellular

   fractionation without using a Dounce homogenizer.  Cells/tissues are passed through a specialized filter

   cartridge.  Plasma membranes of the cells are ruptured during the process and intact nuclei, organelles,

   plasma membrane, and cytosolic proteins are released into a suspensionwhich is further separated into

   five fractions:  total membrane, plasma membrane, cytosolic, organellar, and nucleus fractions.  Due to the

   use of the filter cartridge and a unique buffer system, high yield of native plasma membrane protein can be

   obtained in less than one hour with minimum cross-contaminations.  The native plasma membrane protein

   isolated can be used for any downstream experiments. Unlike all other kits described above, the spin

   column-based plasma membrane isolation kit can alsobe used for plant PM protein isolation. This kit is

   becoming more and more popular as evidenced by the data from selected publications below.

 

The selection of a particular membrane protein isolation kit mainly depends on specific downstream

applications. Some PM isolation kits have much broader applications than others.  For instance, detergent-

free native PM proteins can be used for almost all possible downstream applications, while those isolated by

Pierce cell surface protein isolation kit is only recommended for Western blotting.  Following examples

illustrate what is expected from a good subcellular fractionation kit. Many PM proteins are present in

relatively low concentration.  Sometimes it is difficult to detect and quantify membrane proteins without

isolation and enrichment.  Following data [4] clearly demonstrate the effect of PM protein enrichment on

detectability of SGLT1 from human heart samples.  The SGLT and plasma membrane marker protein Na+/K+

ATPase signals are significantly enhanced in plasma membrane fraction as compared to total membrane

fraction.

 

 

▣ 관련 페이지 ; Invent Biotechologies, Inc.