Choosing the Right Tissue Disruption Technique

When selecting the appropriate tissue disruption technique consider the following characteristics of your experiment:

• Sample Type: What are you lysing? Samples can vary in resilience. For instance soft tissue, such as brain and spleen, is easier to homogenize compared to hard tissue, such as bone. A more aggressive method, such as bead-beating with stainless steel, may be necessary for harder samples.
• Target Molecules: What are you trying to isolate and where is it located? Different methods and/or reagents are needed for nucleic acid isolation versus protein isolation. Similarly, methods for mitochondrial protein isolation versus total protein isolation will vary.
• Required Yield: How much DNA, RNA, or protein do you need to perform the next step? Different methods will result in higher yield, but may be more time-consuming or expensive.
• Intended Downstream Application: What are you doing with the isolated material? Certain reagents commonly used in tissue lysis are incompatible with specific experiments. For instance, if you intend on performing enzymatic assays with your isolated protein of interest, you should choose a method that will retain or restore protein function (i.e. the protein will not be denatured).
• Sample Quantity: Do you need a high-throughput method and/or will you be performing the method frequently? If so, consider investing in specialized equipment that is reliable and efficient, such as the FastPrep^TM Instruments that are optimized for sample preparation for a variety of cells and tissues.
• Budget: What equipment do you already have and how much can you spend? Tissue lysis methods can vary significantly in cost, with do-it-yourself methods being cheaper and high-throughput methods being more expensive. While affordable "home-brew" methods can be sufficient for certain applications and labs, it may require more time spent troubleshooting and optimizing. Consider using ready-to-use buffers and best-in-class bead beating technology to avoid research delays.

Using Physical Disruption to Lyse Tissues

Physical disruption uses equipment, such as beads or mortar and pestle, and high-speed shaking (or manual grinding) to homogenize tissue and extract nucleic acids or proteins. Bead beating methods can range from low to high impaction, breaking down any sample type whether the tissue is hard (e.g. bone) or soft (e.g. brain). It is important to note that physical disruption methods can produce localized heat, particularly when dealing with resilient sample types, so maintaining cold conditions (i.e. cryogenic grinding) may be required to keep target molecules intact.

Physical disruption can range in cost and throughput. Manual grinding with a mortar and pestle is affordable, but time-consuming and often inconsistent across samples. Bead-beating methods range from affordable to expensive, but are consistently effective and can easily be converted to automated systems for high-throughput needs.

Bead beating lysing matrices commonly used for tissue samples to extract nucleic acids and/or proteins:

• Lysing Matrix A: A combination of garnet matrix and large ceramic spheres
• Lysing Matrix D: Ceramic spheres
• Lysing Matrix S: Stainless steel
• Lysing Matrix Z: Yttria-stabilized zirconium oxide beads

Additionally, you can streamline tissue sample preparation using commercially available kits, such as the FastDNA-96 Tissue & Insect DNA Kit.

Liquid-Based Tissue Lysis

Liquid-based cell lysis involves solutions containing detergents or buffers that can weaken or disrupt cell or tissue membranes. Use nonionic (e.g., Triton-X, NP-40) and zwitterionic (e.g., CHAPS) detergents as a mild approach to solubilize membrane proteins while maintaining protein function. Ionic detergents (e.g., sodium deoxycholate, Sodium Dodecyl Sulfate) are strong solubilizing agents and denature proteins, destroying protein activity.

RIPA buffer is a common buffer used to disrupt soft tissue for protein extraction. It is compatible with SDS-PAGE, but not ideal for immunoprecipitation, so dialysis may be required depending on the downstream application to avoid interference with protein interactions. RIPA is a Tris-based solution that contains three detergents: NP40, sodium deoxycholate, and sodium dodecyl sulfate (SDS).

• RIPA buffer recipe: 25 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% NP40, 1% sodium deoxycholate and 0.1% sodium dodecyl sulfate (SDS).

A common alternative to RIPA buffer is using a hypotonic lysis buffer containing a protease and phosphatase inhibitor cocktail to swell the cells, weakening the membrane, to facilitate lysis while retaining protein activity.

Sonication

Hybrid Approach

Quick Tips on How to Optimize Your Lysing Method

Summary

Selecting the appropriate tissue disruption technique is an important step in setting up your experiment for success. There are several factors to consider when choosing between physical disruption, liquid homogenization, and sonication, but oftentimes a hybrid approach is needed.

MP Bio offers a wide range of solutions and resources to meet your tissue homogenization needs.