Lysate Preparation: Why is RIPA Buffer Best for Western Blot
What is in a lysis buffer? Learn the purpose of each component.
Western blotting is a cornerstone of biological research, yet obtaining good results is a frequent challenge for researchers. The quality of the western blot result begins with efficient protein extraction and solubilization from the biological sample, also known as protein lysis. This process involves breaking down the cell and organelle membranes, releasing intracellular proteins without damaging them. Along with using the correct lysis buffer, extracting protein at 4oC minimises degradation, significantly increasing protein yield. For successful protein detection by western blot, the protein must also be denatured (unfolded from its native 3D structure). This is because an antibody only recognises a specific epitope on the protein which might be buried when the protein is folded. There are multiple lysis buffers available depending on the sensitivity and location of the protein of interest, but we have found RIPA buffer to be the best all-rounder.
What Goes into a Lysis Solution?
A lysis solution contains the following components:
1. The buffer system
The pH of the solution is critical. Proteins may precipitate or become unstable when the pH is outside of the physiological range. To avoid this situation, a buffer system such as Tris-HCl is recommended. Besides buffering solutions in this range, a Tris-HCl buffer preserves the physiological ionic strength and prevents the formation of insoluble products with other ions. A HEPES buffering system is another option. We recommend avoiding buffers with high concentrations of potassium, because these can precipitate proteins when sodium dodecyl sulfate (SDS) is present.
2. Salt ions
Salts maintain the ionic strength of the solution which is needed to disrupt the molecular interactions in the sample. An optimal concentration of ionic salts is essential to disrupt cell membrane without damaging proteins of interest. When the salt ion concentration is too high, some proteins may precipitate. Additionally, when ion concentration is too high, a “smiley face” of band migration may result.
3. Chaotropic agents
Chaotropic agents weaken the hydrophobicity of the proteins to solubilize them. There are two kinds of chaotropic agents in a lysis buffer:
a. Urea/thiourea. These molecules unravel hydrophobic regions by disrupting hydrogen bonding between amino acids. Usually when doing protein extraction for a western blot, 6–8M urea and/or 2M thiourea can be used.
b. Detergents. These are a broad class of surfactants. The key to their solubilizing power is their amphiphilic structure. The hydrophobic end binds to the hydrophobic portions of proteins while the hydrophilic end interacts with water, resulting in solubilization.
|SDS (sodium dodecyl sulfate)
|DOC (sodium deoxycholate)
|SLS (sodium lauryl sarcosine)
Note: It is critical in a western blot that the number of negatively charged SDS molecules that bind to a protein is proportional to the protein’s mass, so that the migration rate is influenced only by mass. Adding cationic surfactants in the lysis buffer would disrupt the SDS-protein interaction and make the proteins migrate in the opposite direction.
Due to the complexity of protein biochemistry, it is challenging to predict the optimal surfactant to extract a given protein. Thus, experimenting with different surfactants is recommended if you encounter issues. This is especially recommended for membrane proteins.
4. Protease inhibitors
Tissues and cells often contain large amounts of proteases. During lysis, these are released and, in turn, can digest the target protein. Therefore, protease inhibitors are critical for preserving the target protein. Common protease inhibitors are PMSF (phenylmethylsulfonyl fluoride), Aprotinin, Leupeptin, Pepstatin, and AEBSF-HCL (4-benzenesulfonyl fluoride hydrochloride). PMSF is highly effective and is the most popular choice for lysate preparation.
Many protease inhibitors require a divalent metal ion to function, so a sequestering agent is also often used to inhibit protease activity, such as EDTA. In addition, for phosphorylated target proteins, a phosphatase inhibitor such as sodium fluoride or sodium orthovanadate is needed to preserve the phosphorylated form of the protein. Sodium orthovanadate, in particular, is very effective, but needs to be activated by adjusting the pH of the solution to 10 and then boiling it until the solution is colorless. Other phosphatase inhibitors include sodium pyrophosphate and β-glycerol phosphate.
5. Reducing Agents
Many proteins exist in multimers through disulfide bonds. Reducing agents disrupt these bonds so that the extracted proteins are present in their monomeric form. Common reducing agents are DTT (dithiothreitol) and BME (beta-mercaptoethanol). Reducing agents also minimise oxidation damage caused by cysteine residues.
RIPA Buffer Recipe
Keeping all of this in mind, RIPA buffer is the best choice for sample lysate preparation. We have validated over 13,000 antibodies in WB, and time and time again, experience the best results using RIPA buffer. Over the years we have refined the buffer and below you will find Proteintech’s optimized version:
|For 1000 ml
|50 mM Tris HCl, PH 7.4
|150 mM NaCl
|1% Triton X-100 or NP-40
|0.5% Sodium deoxylcholate
|1 mM EDTA (0.5M stock)
|10 mM Naf
|Add ddH2O to 1000 ml
|Add PMSF to a final concentration of 1mM and any other protease inhibitors immediately before use.
Last updated November 2022
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