Preparing Protein Lysates
Lysate prep is the important first step at the beginning of a western blot experiment. Cell lysis is the breaking down of the cell membrane and the separation of proteins from the non-soluble parts of the cell. Lysate buffers contain different detergents that help to release soluble proteins (Triton-X, Tween, SDS, CHAPS). Dependent on the location of the protein of interest, a different lysate buffer is needed to obtain a high yield and purity of the protein.
However, every protein is different and may react differently with the buffers and detergents. If you don’t get your protein of interest in solution or you are studying a special protein-protein interaction, you can try different buffers and exchange the detergents.
You are watching: Choosing The Right Lysis Buffer
Contents:
- Choosing the right lysis buffer
- Protocol
- Recipes
Choosing the right lysis buffer
Lysate buffers contain different detergents that help to release soluble proteins (Triton-X, Tween, SDS, CHAPS). Dependent on the location of the protein of interest, a different lysate buffer may be needed to obtain a high yield and purity of the protein.
However, every protein is different and may react differently with the buffers and detergents. If you don’t get your protein of interest in solution or you are studying a special protein-protein interaction, you can try different buffers and exchange the detergents.
You are watching: Choosing The Right Lysis Buffer
General Guidelines
The most commonly used buffers are RIPA and NP-40 which are both suitable for whole-cell lysate/membrane bound proteins. RIPA buffer’s harsh properties are best suited for hard to solubilize proteins, which is why it is the preferred choice for nuclear and mitochondrial proteins. A Tris-HCl lysis buffer sometimes shows advantage over RIPA when solubilizing cytoplasmic proteins. Generally, optimal conditions should be tested for the protein of interest.
Location of Protein
Lysis Buffer Recommended
Whole Cell
RIPA or NP-40
Membrane bound
RIPA or NP-40
Nuclear
RIPA*
Mitochondria
RIPA*
Cytoplasmic
Tris-HCl or RIPA
*alternatively for proteins within cellular compartments such as the Nucleus or Mitochondria, fractionation protocols are available which will enrich your sample for these proteins.
Don’t forget your inhibitors!
Denaturation/proteolysis and dephosphorylationen (in case of phosphoproteins) should always be kept to a minimum and added freshly to the cell lysate (EDTA, sodium orthovanadate, PSMF, Aprotinin).
Keeping your lysates stable:
Cell lysates are unstable because the proteins are extracted from their natural environment. For this reason cell lysates should be prepared on ice using pre-cooled buffers and equipment to prevent protein degradation.
Endogenous proteases and phosphatases within the cell are also able to interfere with your lysate, causing proteolysis and dephosphorylation of your sample. This can be kept to a minimum by adding inhibitors to your lysis buffer such as EDTA, PMSF, sodium orthovanadate and Aprotonin.
Protocol – Cell and Tissue Lysate Preparation:
1. Cultured cells:
Pre-cool a refrigerated centrifuge to 4°C. Pellet the cultured cells by centrifugation for 5 minutes at 1000 x g (approximately 2000 rpm) at 4°C. If using adherent cells, remove culture medium, wash with ice-cold 1x PBS and collect cells in 1x PBS using cell scraper, Then pellet by centrifugation for 5 minutes at 1000 x g (approximately 2000rpm at 4°C. Wash 3 times with ice-cold 1X PBS and then add chilled RIPA buffer with protease inhibitor. In general, add 100 μl RIPA buffer for approximately every 106 cells present in the pellet (count cells before centrifugation). Reduce the volume of RIPA buffer accordingly if a higher protein concentration is required.
Vortex to mix and keep on ice for 30 min, vortexing occasionally. Go to step 3,lysis and storage.
2. Tissues:
Dissect the tissue of interest and wash briefly with chilled 1X PBS to remove any blood if necessary, cut the tissue into smaller pieces whilst keeping it on ice. Transfer the tissue to a homogenizer and add RIPA buffer with protease inhibitor. In general, add 500 μl RIPA buffer for approximately every 10 mg of tissue. Homogenize thoroughly and keep the sample on ice for 30 min.
Vortex occasionally. Go to step 3, lysis and storage.
Tip 1: Add phosphatase inhibitors to lysis buffers for extraction of phosphorylated proteins
3. Lysis and Storage
Sonicate the sample to break the cells or tissue up further and to shear DNA. Adjust sonication time to your type of sample: 1 min for cell lysates and 2-5 min for tissue lysates at a power of about 180 watts (in rounds of 10 seconds sonication/10 seconds rest for each cycle). Keep the sample on ice during the sonication.
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Tip 2: The addition of DNase for DNA digestion is not recommended as this introduces protein contamination from the enzyme.
a. Centrifuge at 10,000 x g (approximately 9700 rpm for rotors of a 9.5 cm radius) for 20 minutes at 4°C to pellet cell debris, and then transfer the supernatant to a fresh microfuge tube without disturbing the pellet.
b. Determine protein concentration of the lysate by Bradford or BCA protein assay.
c. Samples can be frozen at -80°C for long-term storage, or be used for immediate Western blotting or immunoprecipitation.
d.For Western blotting, mix sample with 4X SDS sample buffer to a final dilution of 1X. Heat the mixture to 95°C for 5 minutes before loading onto an SDS-PAGE gel.
Recipes:
1x PBS
For 1000mL
10 mM Na₂HPO₄
1.42 g
1.8 mM KH₂PO₄
0.24 g
137 mM NaCl
8 g
2.7 mM KCl
0.2 g
Adjust pH to 7.4
Add ddH₂O to 1000 ml
RIPA Buffer
For 1000mL
50 mM Tris•HCl, pH 7.4
50mL
150 mM NaCl
8.76 g
1% Triton X-100 or NP-40
10mL
0.5% Sodium deoxylcholate
5 g
0.1% SDS
1g
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1 mM EDTA (0.5 M stock)
2mL
10 mM NaF
0.42g
Add ddH₂O to 1000 ml
Add PMSF to a final concentration of 1 mM and any other protease inhibitors immediately before use.
NP-40 buffer
For 1000mL
50mM Tris-HCl, pH 8.5
50mL
150mM NaCl
8.76g
1% NP-40
10mL
Add ddH2O to 1000mL
Add PMSF to a final concentration of 1mM and any other protease inhibitors immediately before use
4x SDS sample buffer
For 1000mL
12% SDS
120g
25% Glycerol
250mL
150 mM Tris•HCl (pH 7.0•1M stock)
150mL
0.03% Bromophenol Blue
300 mg
20% β-mercaptoethanol
200mL
Add ddH₂O to 50 ml, aliquot and store at -20°C
20% β-mercaptoethanol, (or 500 mM DTT replaced), should be added freshly before use.
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