ProteoJET™ Membrane Protein Extraction Kit

This product has been discontinued as of June 15, 2011 and is no longer available. We recommend an alternative product from Thermo Scientific Pierce Protein Research Product line: MEM-PER

- Store at 4°C

Catalog#	Size, concentration	Certificate of Analysis	MSDS

K0321	for 50 preps.	K0321

Product information

Features

High yields of non-denatured, functionally active membrane proteins.
High quality fractionation – no cross-contamination between membrane and cytoplasmic fractions.
Fast and simple protocol.
Compatible with downstream applications:
– protein quantification,
– 1D and 2D electrophoresis,
– immunoprecipitation and Western blotting,
– enzymatic activity and reporter gene assays.

Applications

Extraction of native, non-denatured membrane and cytoplasmic protein fractions from mammalian cultured cells or tissue samples.

Description

The ProteoJET™ Membrane Protein Extraction Kit is designed for efficient isolation of membrane proteins from mammalian cells (suspension and adherent) and tissue samples. The simple and rapid procedure isolates high quality membrane and cytoplasmic protein extracts without cross-contamination between the two fractions. Both fractions contain pure, non-denatured functional proteins which can be used directly in a variety of proteomics applications, such as protein quantification, 1D and 2D electrophoresis, immunoprecipitation and Western blotting, enzymatic activity and reporter gene assays.

The kit is supplied with sufficient reagents for 50 extractions from 5 million cells or 20-40 mg of tissue per experiment.

Quality Control

The kit is functionally tested for membrane and cytoplasmic protein extraction from cultured mammalian suspension cells.

Components

Cell Permeabilization Buffer
Membrane Protein Extraction Buffer
Cell Wash Solution
Detailed Protocol

Storage

All components of the kit should be stored at 4°C.

Figure 1. Membrane proteins extracted using the ProteoJET™ Membrane Protein Extraction Kit from various mammalian samples.
A – from cultured cells (adhesion and suspension)
M – PageRuler™ Plus Prestained Protein Ladder
1 – Jurkat cells
2 – HeLa suspension cells
3 – HeLa cells
4 – NIH3T3 cells
5 – HEK293 cells
B – from mouse tissues
M – PageRuler™ Plus Prestained Protein Ladder
1 – liver
2 – kidney
3 – spleen
4 – lung
5 – muscle
6 – brain

$High quality extraction and fractionation of membrane (M) and cytoplasmic (C) proteins using the ProteoJET™ Membrane Protein Extraction Kit$

Figure 2. High quality extraction and fractionation of membrane (M) and cytoplasmic (C) proteins using the ProteoJET™ Membrane Protein Extraction Kit compared to competing kits from two other vendors.
HeLa cells were transfected with two plasmids encoding Green fluorescent protein localized in the cytoplasm and Red fluorescent protein localized in the membrane. Protein extraction was followed by native electrophoresis. The ProteoJET™ Membrane Protein Extraction Kit yields cytoplasmic and membrane fractions with virtually no cross-contamination while extraction performed with competing kits produced substantial cross-contamination between fractions.

Figure 3. Enzymatic activity of membrane and cytoplasmic proteins extracted using the ProteoJET™ Membrane Protein Extraction Kit and a competing kit from another vendor.
The activity of proteins in the cytoplasmic and membrane fraction demonstrates both the high quality fractionation and functional activity of proteins extracted with the ProteoJET™ Membrane Protein Extraction Kit.
C – cytoplasmic proteins
M – membrane proteins

$Western blot analysis of membrane and cytoplasmic protein fractions extracted using the ProteoJET™ Membrane Protein Extraction Kit$

Figure 4. Western blot analysis of membrane and cytoplasmic protein fractions extracted using the ProteoJET™ Membrane Protein Extraction Kit and a competing kit from another vendor.
Antibodies specific for membrane proteins Pan-Cadherin and Calnexin and an antibody specific for the cytoplasmic protein Tubulin were used to evaluate the quality of protein fractions. Western blot analysis of proteins extracted using the ProteoJET™ Membrane Protein Extraction Kit demonstrates the presence of cytoplasmic and membrane proteins in the correct fraction without cross-contamination between fractions. Western blot analysis of proteins extracted with the competitor kit demonstrates poor fractionation with substantial cross-contamination between fractions.
T – total protein
C – cytoplasmic protein fraction
M – membrane protein fraction

Patents, Licenses, Trademarks

Protocols & recommendations

RECOMMENDATIONS FOR USE

ADDITIONAL PROTOCOLS

Protein Samples: Extraction and Quantification Guides

Sample/procedure	Extraction	Quantification
Mammalian cell and tissue samples	Extract total proteins using ProteoJET™ Mammalian Cell Lysis Reagent. Extract nuclear and cytoplasmic proteins using ProteoJET™ Cytoplasmic and Nuclear Protein Extraction Kit.	Use Bradford Reagent, ready-to-use for protein quantification. Easily create standard curves by using one of our convenient protein standard sets: Bovine Serum Albumin Standard Set, Bovine Gamma Globulin Standard Set, Protein Standard Solution. Create a standard curve by plotting the 595 nm values (y-axis) versus their concentration in µg/ml (x-axis). Use the standard curve to determine the protein concentration of each unknown sample.
Bacterial samples	To analyze total bacterial proteins in SDS-PAGE, cells can be treated directly with DualColor™ Protein Loading Buffer Pack or Protein Loading Buffer Pack.
Lyophilized proteins	To analyze proteins in SDS-PAGE, treat lyophilized proteins directly with DualColor™ Protein Loading Buffer Pack or Protein Loading Buffer Pack.

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