Cell Lysate: Overview & Applications

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Overview: Cell Lysate

Cell lysate is the preparation obtained after lysing a cell population in labs using certain chemical reagents and enzymes, or by osmotic and mechanical disruption. 

They can be generated either from whole cells (called whole cell lysate) containing cell membrane, and cytoplasmic and nuclear proteins, or nuclear extracts, which mainly contain nuclear proteins. 

The cell lysate has applications in the purification of whole organelles, nucleic acids (genomic DNA and RNA), and cellular proteins. Additionally, lysates of cell lines, containing tagged or untagged overexpressed proteins, are also used as a positive control to study antibody reactivity in western blots and ELISA.

The preparation of cell lysate by cell lysis is a routine in vitro workflow in molecular biology labs. It provides crucial insights into the cellular reactions, components, and functioning inside the cells.

Today, specific cell lysates are prepared and a spectrum of control cell lysates are commercially available to assist researchers with their experiments. Furthermore, there are several techniques available for prepping lysates depending on their cell type, such as fungal, bacterial, or mammalian cells.

In this article, we will cover cell lysate preparation methods, the experiments involving cell lysates, and the lab areas that extensively use them on a routine basis.

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How Are Cell Lysates Prepared?

Cells are the fundamental units of living organisms. They contain organelles that facilitate diverse cell functions and nucleic acids that store genetic information for the development and function of organisms. 

The cellular contents are surrounded by a cell membrane. Thus, it needs to be broken to access the information coded in organisms’ DNA and RNA sequences for research and medical purposes. This is done by the process called cell lysis.

What is Cell Lysis?

Cell lysis is a method to break open the cell using enzymatic, chemical, and mechanical procedures. The released fluid contains the content of lysed cells such as proteins and nucleic acids. 

Furthermore, to prevent the modification of the macromolecules of the cells, protease and phosphatase inhibitors are added to the lysis buffer. Then, the purified lysates are used in further research studies.

Cell Lysis Methods

There are multiple ways to lyse the cell and prepare cell lysates. They are mainly characterized into two groups:

  • Mechanical Methods– Mechanical lysis is a high-throughput method in which cells are broken physically by shear force. It’s of two types:
    • High-pressure Homogenizer: In this method, cell membranes are broken by forcing the cell to move through an orifice valve using high pressure. It’s used for large-scale microbial disruption.
    • Bead Mill: In this method, the cell suspension is mixed with tiny beads made of ceramic, glass, and steel, and agitating the beads at high speed to disrupt the cells. 
  • Non-Mechanical Method– Non-mechanical lysis is disrupting the cells using certain compounds and enzymes, rather than using shear force. They are characterized into three groups based on the method they utilize:
    • Physical: In this method, cells are broken using different forces such as heat, pressure, and sound energy. It includes methods like:
      • Cavitation: Involves the formation and rupture of cavities and bubbles using sonication or increased velocities.
      • Thermolysis: Involves repeated freeze/thaw cycles of cells to disrupt cell membranes and prepare lysate.
      • Osmotic Shock: Cells are disrupted by changing salt concentration surrounding them. 
    • Chemical: The disruption of a cell membrane using a lysis buffer containing certain chemicals. It’s of three types:
      • Detergent Lysis: Utilizes surfactants that break hydrophobic and hydrophilic interaction of the cell membrane, releasing the cellular component. Other than this, chaotropic agents are also used, such as Ethylenediaminetetraacetic acid (EDTA), guanidine, and urea.
      • Alkaline Lysis: Here, OH_ (sodium hydroxide)  is the main component used in lysis buffers to lyse cells. The ion reacts with the component of the cell membrane and breaks the fatty acid-glycerol ester bond to release cellular components.
    • Enzymatic: In this method, enzymes such as cellulose, protease, lysozyme, zymolyase, lysostaphin, or glycanase, are used for cell lysis procedures based on the cells.

What Are Cell Lysates Used For?

The purified cell lysates are used in a range of life science labs, including molecular biology, biochemistry, and cell biology laboratory, in sample preparation for lab assays. They are prepared by lysing the cell cultures and purifying the components using procedures, such as protein purification, and DNA and RNA extraction.

The prepared lysates are used in: 

  • Immunoassays for point-of-care diagnostics.
  • Understanding the cellular heterogeneity in cell cultures.
  • Transcriptomics, genomics, proteomics, and metabolomics studies.
  • Molecular diagnostics of pathogens.
  • Down streaming processes such as mRNA transcriptome determination, protein purification, drug screening, cancer diagnostics, and analysis of the composition of specific proteins, lipids, and nucleic acids individually or as complexes.

Here are lab workflows that utilize cell lysates to achieve specific purposes:

Western Blotting

In western blotting, detergent-based or physical lysing methods are used to prepare cell lysates. The preparation of lysate for the assays differs based on the cell types, such as adherent cells and suspension cultures. Thus, refer to proper guidelines for each assay.

Given below is a general procedure to prepare total cell lysate for the workflow:

  1. Solubilize cells (approximately 2×106-1×107 cells per mL) in sample buffer such as 2X SDS sample buffer (6% SDS, 10% glycerol, 20 mM dithiothreitol, 0.25 M Tris, 10 mM NaF and bromophenol blue).
  2. Adjust the pH to 6.8.
  3. In a boiling water bath, heat the extracts for five minutes.
  4. Sonicate the extract 3-4 times for 5-10 seconds.
  5. Measure protein concentration in the purified lysate using Lowry, BCA, or Bradford assays, before loading on a gel.

SDS-Page

The detergent-based lysis method is the most preferable technique to prepare lysates for gel electrophoresis. Here’s a procedure for preparing whole cell lysate for SDS-PAGE:

  1. Wash cell sin PBS and then incubate them in RIPA buffer containing 150 mM sodium chloride, 1 mM EDTA, 1.0% NP-40, 50 mM Tris HCl, pH 7.4, 0.1% SDS, 0.5% sodium deoxycholic acid, and 0.01% (w/v) sodium azide for cell lysis.
  2. Use and protease inhibitors, specific for the inhibition of cysteine, aspartic, and serine proteases to ensure protein integrity. Also, add phosphatase inhibitors.
  3. Remove cell debris using the centrifugation technique.
  4. Perform optimal dilution of the lysate and measure the protein concentration through suitable protein assay, such as BCA.

ELISA

ELISA is short for enzyme-linked immunosorbent assay. It’s an analytical biochemistry assay used to detect antibodies in biological samples. Below is a procedure to prepare cell lysates for ELISA workflow:

  1. Solubilize cells in lysis buffer.
  2. Let the cell sit on ice for 30 minutes.
  3. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble materials.
  4. In a new tube, aliquot the supernatant and discard the remaining cell extract.
  5. Determine protein concentration using a total protein assay.

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Cell lysates are preparations made by lysing cells using physical, chemical, and enzymatic methods. They are extensively used in molecular biology and biotech labs in a range of workflows, including ELISA, western blotting, and SDS-PAGE.

Each assay or workflow follows a specific protocol for cell lysate preparations. Thus, the optimization of protocols to prepare cell lysates for specific workflows should be done to ensure the quality and reliability of assay data/results. 

Apart from the optimized protocol, the quality of reagents and equipment also impacts your research quality.

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