Choosing The Right Lysis Buffer
リンク
- Western Blot Protocol
- How To Optimize Your Western Blot
- SDS-PAGE Gel Recipes
- How To Optimize Your Results With Low MW Proteins
- Tricine Gel Recipe For Low Molecular Weight Proteins
- Choosing The Right Lysis Buffer
- Choosing The Right Western Blot Detection Method
- Western Blot Troubleshooting: Why Does The Observed Protein Molecular Weight (MW) Differ From The Calculated One?
- Western Blot Troubleshooting: High Background
- Western Blot Troubleshooting: Weak/No Signal & Other
- Western Blot ppt
- Western Blot Video Protocol
Preparing Protein Lysates
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.
General Guidelines
Whole-cell lysate/membrane-bound proteins
The most commonly used buffers are RIPA and NP-40. RIPA buffer’s harsh properties are best suited for hard to-solubilize proteins.
RIPA: 25mM Tris, HCl (pH 7.6) 150mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS
NP-40: 50 mM Tris, HCl (pH 8.5) 150 mM NaCl, 1% detergent
Nuclear/mitochondria proteins
RIPA is the preferred choice here. However, fractions protocols are often used to increase the concentration of the desired protein.
Cytoplasmic proteins
A Tris-HCl lysis sometimes shows advantages over RIPA buffer. Optimal conditions should be tested for the protein of interest.
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).
