C buffer, A x 2

C buffer, A x 2
Compartmental Protein Extraction Kit K3013010 or K3012010 User Manual
Biochain Institute, Inc.
Compartmental Protein Extraction Kits
Extracting CNMCS (Cytoplasmic, Nuclear, Membrane, and Cytoskeleton) proteins in one kit
Introduction
One of the challenges of functional proteomics is separation of complex protein mixtures for
quantitative and differential subcellular localization analysis. This necessitates standardized and
repeatable operation procedures to isolate subcellular proteomes from tissues and cells.
BioChain’s CNMCS and CNM Compartmental Protein Extraction Kits address the challenge by
providing an innovative, easy-to-perform, and cost-effective method to sequentially isolate
cytoplasmic, nuclear, membrane, and cytoskeleton proteins from mammalian tissues and cells
based on a proprietary technique. These unique kits can isolate four or three compartmental
proteins. They are excellent tools for the initial purification and preparation of proteins for
downstream applications including SDS-PAGE, Western blotting, gel mobility shift, protein assays
and other procedures.
Kd
M
1
2
3
4
5
250
130
95
72
55
36
28
17
11
GAPDH
Example of CNMCS Protein Extraction from
Mammalian Tissue
Total Protein and compartmental proteins extracted
from rat colon tissue using Biochain’s Total Protein
Extraction Kit and CNMCS Compartmental Protein
Extraction Kit were submitted to SDS-PAGE in 5
identical gels. Coomassie staining of one piece of
gel indicated distinct protein pattern of respective
fractions. Immunoblotting of PVDF membranes
transferred from 5 other pieces of gel against
cytoplasmic marker protein GAPDH, nuclear marker
protein Histone H1, membrane marker protein
+
+
Na /K ATPase, and cytoskeleton marker protein
vimentin assigned the majorities of the marker
proteins to their expected compartmental fractions.
Lane 1: Total Protein; Lane 2: Cytoplasmic Protein;
Lane 3: Nuclear Protein; Lane 4: Membrane Protein;
Lane 5: Cytoskeleton Protein.
Histone
Na+/K+ ATPase
Vimentin
Features
• Convenient - Provides a complete set of components for stepwise preparation of
cytoplasmic, nuclear, and membrane proteins from mammalian tissues and cultured cells
• Fast – isolate four or three compartmental proteins in less than three hours
• Reliable - Super-quality and highly reproducible separation of subcellular proteome
fractions as verified by clearly distinct protein patterns and precise subcellular
localizations of marker proteins
• Pure - Minimal cross contaminations
• Simple - Easy to use comparing to other methods, such as differential centrifugations
Applications
• Detection of differential post-translational modifications or differential subcellular
localization of target proteins
• Enrichment of low-abundance proteins for visualization and subsequent analysis
BioChain Institute, Inc. 3517 Breakwater Avenue, Hayward, CA 94545
Tel: 888-762-2568 Fax: 510-783-5386 E-mail: [email protected] Website: www.biochain.com
1
Compartmental Protein Extraction Kit K3013010 or K3012010 User Manual
•
•
•
Biochain Institute, Inc.
Preparation of nuclear extract from mammalian tissues and cultured cells is crucial for
studying DNA binding proteins such as transcription factors employing gel mobility shift
techniques
Preparations of cytoplasmic fractions are useful to study soluble proteins abundant in
cytosol
Preparations of membrane fractions to study membrane proteins such as receptors
Description
Components in this kit are prepared with pure chemicals according to the proprietary technology.
To prevent protein degradation, a ready-to-use protease inhibitor cocktail is provided. BioChain’s
Compartment Protein Extraction Kits are designed to sequentially isolate cytoplasmic, nuclear,
membrane, and cytoskeleton proteins from mammalian tissues and cells. One Kit is consisted of
reagents enough to enrich four or three compartmental proteins from 5 grams tissues or about
125 million cells. The efficiency of subcellular fractionation has been investigated by SDS-PAGE
and immunoblotting of selected marker proteins.
Quality Control
One kit of this lot has been tested to go through the complete compartment protein extraction
procedure from rat colon. Four compartmental proteins from the extraction are used for
electrophoresis, transferring to PVDF membrane and immunoblotting with Mouse anti-GAPDH,
Mouse anti-Histone H1, Mouse anti-Na+/K+ ATPase, and Mouse anti-Vimentin as primary
antibodies, and HRP-conjugated anti-Mouse IgG as secondary antibody. The separation of four
compartmental proteins is confirmed by the enrichment of GAPDH in the cytoplasmic fraction,
Histone H1 in the nuclear fraction, Na+/K+ ATPase in the membrane fraction, and the unique
localization of Vimentin in the cytoskeleton fraction.
Contents
CNMCS Kit
Item
Buffer C
Buffer W
Buffer N
Buffer M
Buffer CS
50 x PI
Component
Amount
Part No.
HEPES (pH7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium
OrthoVanadate
HEPES (pH7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium
OrthoVanadate
HEPES (pH7.9), MgCl2, NaCl, EDTA, Glycerol, Sodium OrthoVanadate
HEPES (pH7.9), MgCl2, KCl, EDTA, Sucrose, Glycerol, Sodium
deoxycholate, NP-40, Sodium OrthoVanadate
Pipes (pH6.8), MgCl2, NaCl, EDTA, Sucrose, SDS, Sodium
OrthoVanadate
A cocktail of protease inhibitors
18 ml
K3013010-1
50 ml
K3013010-2
6 ml
6 ml
K3013010-3
K3013010-4
3 ml
K3013010-5
0.66 ml
K3013010-6
CNM Kit
Item
Amount
Part No.
Buffer C
12 ml
K3012010-1
Buffer W
50 ml
K3012010-2
Buffer N
6 ml
K3012010-3
Buffer M
6 ml
K3012010-4
50 x PI
0.5 ml
K3012010-5
Reagents are sufficient for extraction of compartment proteins from 5 g tissue or about
125 million cells. Minimal 0.1 g tissue or 2.5 million cells should be required.
Storage and Stability
Buffer C, W, N, M, and CS are stored at 2-8 °C. The 50 x PI solution should be stored at
-20 °C. If the kit is going to be used for multiple extractions, aliquot the PI solution properly
before storing. The kit is stable for 1 year when handled properly.
Reagents and Equipments not provided
• Tissue Homogenizer
• Centrifuge and microcentifuge
• Rolling facility
BioChain Institute, Inc. 3517 Breakwater Avenue, Hayward, CA 94545
Tel: 888-762-2568 Fax: 510-783-5386 E-mail: [email protected] Website: www.biochain.com
2
Compartmental Protein Extraction Kit K3013010 or K3012010 User Manual
Biochain Institute, Inc.
Reference
1. Neelam Sharma-Walia et al J. Virol., Apr 2004; 78: 4207 – 4223
2. Christian Korn et al J. Biol. Chem., Nov 2004; 10.1074/jbc.M413035200
Protocol
I. Extracting compartment proteins extraction from tissues.
For buffer C, N, M, and CS, add 50xPI to working concentration (1 x) before usage.
1. Weigh certain amount of tissue (Wt gram), chop it to small pieces, then pipette ice cold
buffer C at 2.0 ml per gram tissue. Homogenize* the tissue at moderate speed (e.g.,
speed 4) for 20 sec. Let it stand on ice for a few seconds, repeat homogenization twice.
2. Rotate the mixture at 4˚C for 20 min. Spin at 18,000 g at 4˚C for 20 min. The
cytoplasmic proteins are in the supernatant, take out and save in another tube.
3. Resuspend the pellet with Wt x 4 ml ice cold buffer W, rotate at 4˚C for 5' min. Spin at
18,000 g at 4˚C for 20 min. Drain the supernatant.
4. Add Wt x 1.0 ml ice cold buffer N to resuspend pellet from step 3, rotate at 4˚C for 20
min. Spin at 18,000 g at 4˚C for 20 min. The nuclear proteins** are in the supernatant,
take out and save it in another tube.
5. Add Wt x 1.0 ml ice cold buffer M to resuspend pellet from step 4, rotate at 4˚C for 20
min. Spin at 18,000 g at 4˚C for 20 min. The membrane proteins are in the supernatant.
Take out and save in another tube.
6. Pre-warm CS buffer at RT or 37 ˚C to make it clear. Add Wt x 0.5 ml buffer CS to
resuspend pellet from step 5, rotate at room temperature for 20 min. Spin at 18,000 g at
4˚C for 20 min, save supernatant.
7. Resuspend the pellet from step 6 with Wt x 1.5 ml ice cold buffer C, rotate at 4˚C for 5
min. Spin at 18,000 g at 4˚C for 20 min, save supernatant.
8. Combine the supernatants from step 6 and 7. The cytoskeleton proteins are in the mixed
solution.
9. Measure the protein concentrations of four fractions. Aliquot and label the proteins
properly, store at –70˚C.
* We recommend using IKA Ultra Turrax T25 Basic or similar model homogenizer for
tissues. Manual homogenizer can also be used; the purpose of the homogenization is to
get the tissue lysed completely without breaking the nuclear.
** A dialysis step may be necessary if the nuclear fraction is going to be used for the gel
mobility shift assay
II. Extracting compartment proteins from culture cells
For buffer C, N, M, and CS, add 50xPI to working concentration (1 x) before usage.
1. Pellet the culture cells using routine cell culture techniques. Count cells and add ice cold
buffer C at 2.0 ml per 20 million cells. Mix the cells with buffer C well and rotate the
mixture at 4˚C for 20 min.
2. Prepare a syringe with a needle gauged between 26.5 and 30. Remove the needle tip by
bending the needle several times and only leave the needle base on the syringe. Pass
the cell mixture through needle base 50-90 times to disrupt the cell membrane and
release the nuclei from the cells. The degree of cell membrane disruption and nucleus
release can be monitored under microscope. 90-95% of the nuclei should be released
from the cells. Then spin the cells mixture at 15,000 g at 4˚C for 20 min. The cytoplasmic
proteins are in the supernatant, take out and save in another tube.
3. Resuspend the pellet with ice cold buffer W at 4.0 ml per 20 million cells, rotate at 4˚C
for 5 min. Spin at 15,000 g at 4˚C for 20 min. Drain the supernatant.
BioChain Institute, Inc. 3517 Breakwater Avenue, Hayward, CA 94545
Tel: 888-762-2568 Fax: 510-783-5386 E-mail: [email protected] Website: www.biochain.com
3
Compartmental Protein Extraction Kit K3013010 or K3012010 User Manual
Biochain Institute, Inc.
4. Add ice cold buffer N at 1.0 ml per 20 million cells to resuspend pellet from step 3,
rotate at 4˚C for 20 min. Spin at 15,000 g at 4˚C for 20 min. The nuclear proteins are in
the supernatant, take out and save it in another tube.
5. Add ice cold buffer M at 1.0 ml per 20 million cells to resuspend pellet from step 4,
rotate at 4˚C for 20 min. Spin at 15,000 g at 4˚C for 20 min. The membrane proteins are
in the supernatant. Take out and save in another tube.
6. Pre-warm CS buffer at RT or 37 ˚C to make it clear. Add buffer CS at 0.5 ml per 20
million cells to resuspend pellet from step 5, rotate at room temperature for 20 min.
Spin at 15,000 g at 4˚C for 20 min, save supernatant.
7. Measure the protein concentrations of four fractions. Aliquot and label the proteins
properly, store at –70˚C.
Trouble Shooting
1. Cytoplasmic proteins-carryover to nuclear and membrane fractions
The tissue is not completely homogenized. Increase the speed and times of homogenization.
For protein extraction from cells, increase the needle gauge size and increase the times of
cells passing the needle base. Also repeat the washing step (step 3 in the protocol) 1-2 times
to completely remove cytoplasmic proteins. Add a washing step right after nuclear protein
extraction.
2. Nuclear proteins present in the cytoplasmic fraction
The tissue homogenization condition was too harsh, and the cytoplasmic membrane and
nuclear membrane were broken. Decrease the speed and times of homogenization. For
protein extraction from cells, decrease the needle gauge size and decrease the times of cells
passing the needle base.
3. Nuclear proteins present in the membrane fraction
Repeat the nuclear proteins-extraction step 1-2 times before membrane proteins-extraction
4. Membrane proteins present in the cytoplasmic and nuclear fractions
The tissue homogenization condition or the cell disruption condition was too harsh, and the
nuclear membrane was broken.
5. Protein degradations
Make sure PI was added to Buffers C, N, M, and CS before use
50 x PI was not aliquot, and lost activity when frozen-thawed for too many times.
Homogenize the tissue for less time (< 20 sec); let the tube stand on ice for a few seconds
before next homogenization
Related Products
Compartmental proteins, Total Proteins, Total Protein Extraction Kit
BioChain Institute, Inc. 3517 Breakwater Avenue, Hayward, CA 94545
Tel: 888-762-2568 Fax: 510-783-5386 E-mail: [email protected] Website: www.biochain.com
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