Lysis buffer

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A lysis buffer is a buffer solution used for the purpose of breaking open cells for use in molecular biology experiments that analyze the labile macromolecules of the cells (e.g. western blot for protein, or for DNA extraction). Most lysis buffers contain buffering salts (e.g. Tris-HCl) and ionic salts (e.g. NaCl) to regulate the pH and osmolarity of the lysate. Sometimes detergents (such as Triton X-100 or SDS) are added to break up membrane structures. For lysis buffers targeted at protein extraction, protease inhibitors are often included, and in difficult cases may be almost required. Lysis buffers can be used on both animal and plant tissue cells. [1]

Contents

Choosing a buffer

The primary purpose of lysis buffer is isolating the molecules of interest and keeping them in a stable environment. For proteins, for some experiments, the target proteins should be completely denatured, while in some other experiments the target protein should remain folded and functional. Different proteins also have different properties and are found in different cellular environments. Thus, it is essential to choose the best buffer based on the purpose and design of the experiments. The important factors to be considered are: pH, ionic strength, usage of detergent, protease inhibitors to prevent proteolytic processes. [2] For example, detergent addition is necessary when lysing Gram-negative bacteria, but not for Gram-positive bacteria. [3] It is common that a protease inhibitor is added to lysis buffer, along with other enzyme inhibitors of choice, such as a phosphatase inhibitor when studying proteins with phosphorylation.

Components

Buffer

Buffer creates an environment for isolated proteins. Each buffer choice has a specific pH range, so the buffer should be chosen based on whether the experiment's target protein is stable under a certain pH. Also, for buffers with similar pH ranges, it is important to consider whether the buffer is compatible with the experiment's target protein. [4] The table below contains several most commonly used buffers and their pH ranges. [4]

BufferpH Range
Sodium dihydrogen phosphate / disodium hydrogen phosphate5.8 - 8.0
Tris - HCl7.0 - 9.0
HEPES - NaOH7.2 - 8.2

Additives

Salts

Lysis buffer usually contains one or more salts. The function of salts in lysis buffer is to establish an ionic strength in the buffer solution. Some of the most commonly used salts are NaCl, KCl, and (NH4)2SO4. They are usually used with a concentration between 50 and 150 mM. [4]

Sodium dodecyl sulfate (SDS) structure SDS with structure description.svg
Sodium dodecyl sulfate (SDS) structure

Detergent

Triton X-100 structure Triton X-100 groups.svg
Triton X-100 structure

Detergents are organic amphipathic (with hydrophobic tail and a hydrophilic head) surfactants. They are used to separate membrane proteins from membrane because the hydrophobic part of detergent can surround biological membranes and thus isolate membrane proteins from membranes. [5] Although detergents are widely used and have similar functions, the physical and chemical properties of detergents of interest must be considered in light of the goals of an experiment.

Detergents are often categorized as nonionic, anionic, cationic, or zwitterionic, based on their hydrophilic head group feature. [5]

Nonionic detergents like Triton X-100 and zwitterionic detergents like CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate) are nondenaturing (will not disrupt protein functions). Ionic detergents like sodium dodecyl sulfate (SDS) and cationic detergents like ethyl trimethyl ammonium bromide are denaturing (will disrupt protein functions). [6] Detergents are a major ingredient that determines the lysis strength of a given lysis buffer.

Detergent-Free Cell Lysis Buffers

One common issue faced by many cell lysis buffers is the disruption of protein structures during the lysis process, partially caused by use of detergents. Detergents often prevent the restoration of native conditions necessary for proper protein folding. [7]

For the longest time, after a detergent-based cell lysis, a buffer exchange and/or dialysis had to be performed to remove the detergent among other hindering compounds to restore native conditions. [8]

To overcome this a solution has emerged in the form of a detergent-free cell lysis buffer. The GentleLys buffer employs copolymers instead of detergents, ensuring efficient cell lysis while maintaining the native environment crucial for the correct folding of cellular components, such as proteins.

Others

Other additives include metal ions, sugar like glucose, glycerol, metal chelators (e.g. EDTA), and reducing agents like dithiothreitol (DTT). [4]

Commonly used buffers

NP-40 lysis buffer

It may be the most widely used lysis buffer. The solubilizing agent is NP-40, which can be replaced by other detergents at different concentrations. Since NP-40 is a nonionic detergent, this lysis buffer has a milder effect than RIPA buffer. It can be used when protein functions are to be retained with minimal disruption. [9]

Recipe: [9]

RIPA (RadioImmunoPrecipitation Assay) lysis buffer

RIPA buffer is a commonly used lysis buffer for immunoprecipitation and general protein extraction from cells and tissues. The buffer can be stored without vanadate at 4 °C for up to 1 year. [10] RIPA buffer releases proteins from cells as well as disrupts most weak interactions between proteins. [9]

Recipe: [10]

SDS (sodium dodecyl sulfate) lysis buffer

SDS is ionic denaturing detergent. Hot SDS buffer is often used when the proteins need to be completely solubilized and denatured.

Recipe: [10]

ACK (Ammonium-Chloride-Potassium) lysing buffer

ACK is used for lysis of red blood cells in biological samples where other cells such as white blood cells are of greater interest. [11]

Recipe: [12] [13]

GentleLys (Gentle Lysis)

The GentleLys buffer employs synthetic nanodisc copolymers to gently disrupt the cell membrane, offering a milder alternative to conventional detergent-based lysis buffers. This gentle approach eliminates the need for harsh chemicals, creating an environment that preserves the native state of cellular proteins. Consequently, the proteins maintain their structural integrity and functionality, a marked departure from the denaturing effects of detergent-based buffers.

Detergents, salts & enzymes

Cell lysis is a critical step in the purification of enzymes from bacterial cells, various components are commonly included in lysing buffers to facilitate effective cell disruption and release of the target enzyme. These components include detergents, salts, and enzymes, each playing a specific role in the lysis process. Examples of detergents used in lysing buffers include:

Detergents:

Detergents are amphipathic molecules that possess both hydrophilic and hydrophobic properties. In the context of cell lysis, detergents act by disrupting the lipid bilayer of the bacterial cell membrane, leading to membrane permeabilization and release of intracellular components, including the target enzyme.

Commonly used detergents in lysing buffers include:

a. Triton X-100: a nonionic detergent frequently employed due to its mild and effective membrane-disrupting properties, it solubilizes lipids and membrane proteins, allowing the release of intracellular contents.

b. Sodium dodecyl sulfate (SDS): an anionic detergent that denatures proteins by disrupting their secondary and tertiary structures, it solubilizes cellular membranes and aids in protein extraction.

c. Tween-20: a nonionic detergent is milder compared to SDS and Triton X-100. It assists in membrane permeabilization and solubilization of proteins without causing significant denaturation.

Salts:

Salts are crucial components of lysing buffers as they help maintain optimal cellular conditions and provide ionic strength to facilitate cell disruption.

Commonly used salts in lysing buffers include:

a. Sodium chloride (NaCl): NaCl is often included to maintain isotonic conditions, preventing osmotic shock and cell rupture during the lysis process.

b. Potassium chloride (KCl): Similar to NaCl, KCl can be used to adjust the ionic strength and facilitate cell lysis.

Enzymes:

Certain enzymes are added to lysing buffers to enhance cell lysis by digesting specific cellular components that can interfere with the extraction of the target enzyme.

Examples of enzymes used in lysing buffers include:

a. Lysozyme: Lysozyme breaks down the peptidoglycan layer of bacterial cell walls, weakening their structural integrity and facilitating subsequent disruption. It is particularly effective for Gram-positive bacteria.

b. DNase (Deoxyribonuclease): DNase degrades DNA present in the lysate, reducing its viscosity and preventing DNA-related interference in downstream purification steps.

c. RNase (Ribonuclease): Similar to DNase, RNase degrades RNA in the lysate, reducing its viscosity and minimizing RNA-related interference.

The specific combination and concentrations of detergents, salts, and enzymes in lysing buffers can vary depending on the target enzyme, cell type, and experimental requirements, optimization of these components is crucial to achieve efficient cell lysis while preserving the stability and activity of the desired enzyme during the purification process.

Lysis buffer in DNA and RNA studies

In studies like DNA fingerprinting the lysis buffer is used for DNA isolation. Dish soap can be used in a pinch to break down the cell and nuclear membranes, allowing the DNA to be released. Other such lysis buffers include the proprietary Qiagen product Buffer P2.

Related Research Articles

Sodium dodecyl sulfate (SDS) or sodium lauryl sulfate (SLS), sometimes written sodium laurilsulfate, is an organic compound with the formula CH3(CH2)11OSO3Na and structure H3C−(CH2)11−O−S(=O)2−O−Na+. It is an anionic surfactant used in many cleaning and hygiene products. This compound is the sodium salt of the 12-carbon organosulfate. Its hydrocarbon tail combined with a polar "headgroup" give the compound amphiphilic properties that make it useful as a detergent. SDS is also component of mixtures produced from inexpensive coconut and palm oils. SDS is a common component of many domestic cleaning, personal hygiene and cosmetic, pharmaceutical, and food products, as well as of industrial and commercial cleaning and product formulations.

<span class="mw-page-title-main">Detergent</span> Surfactants with cleansing properties

A detergent is a surfactant or a mixture of surfactants with cleansing properties when in dilute solutions. There are a large variety of detergents, a common family being the alkylbenzene sulfonates, which are soap-like compounds that are more soluble in hard water, because the polar sulfonate is less likely than the polar carboxylate to bind to calcium and other ions found in hard water.

<span class="mw-page-title-main">Polyacrylamide gel electrophoresis</span> Analytical technique

Polyacrylamide gel electrophoresis (PAGE) is a technique widely used in biochemistry, forensic chemistry, genetics, molecular biology and biotechnology to separate biological macromolecules, usually proteins or nucleic acids, according to their electrophoretic mobility. Electrophoretic mobility is a function of the length, conformation, and charge of the molecule. Polyacrylamide gel electrophoresis is a powerful tool used to analyze RNA samples. When polyacrylamide gel is denatured after electrophoresis, it provides information on the sample composition of the RNA species.

<span class="mw-page-title-main">Surfactant</span> Substance that lowers the surface tension between a liquid and another material

Surfactants are chemical compounds that decrease the surface tension or interfacial tension between two liquids, a liquid and a gas, or a liquid and a solid. The word "surfactant" is a blend of surface-active agent, coined in 1950. As they consist of a water-repellent and a water-attracting part, they enable water and oil to mix; they can form foam and facilitate the detachment of dirt.

<span class="mw-page-title-main">Western blot</span> Analytical technique used in molecular biology

The western blot, or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detecting the proteins, this technique is also utilized to visualize, distinguish, and quantify the different proteins in a complicated protein combination.

Lysis is the breaking down of the membrane of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a lysate. In molecular biology, biochemistry, and cell biology laboratories, cell cultures may be subjected to lysis in the process of purifying their components, as in protein purification, DNA extraction, RNA extraction, or in purifying organelles.

Protein purification is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Protein purification is vital for the specification of the function, structure and interactions of the protein of interest. The purification process may separate the protein and non-protein parts of the mixture, and finally separate the desired protein from all other proteins. Ideally, to study a protein of interest, it must be separated from other components of the cell so that contaminants will not interfere in the examination of the protein of interest's structure and function. Separation of one protein from all others is typically the most laborious aspect of protein purification. Separation steps usually exploit differences in protein size, physico-chemical properties, binding affinity and biological activity. The pure result may be termed protein isolate.

<i>n</i>-Octyl β-<small>D</small>-thioglucopyranoside Chemical compound

n-Octyl β-d-thioglucopyranoside is a mild nonionic detergent that is used for cell lysis or to solubilise membrane proteins without denaturing them. This is particularly of use in order to crystallise them or to reconstitute them into lipid bilayers. It has a critical micelle concentration of 9 mM.

The first isolation of deoxyribonucleic acid (DNA) was done in 1869 by Friedrich Miescher. DNA extraction is the process of isolating DNA from the cells of an organism isolated from a sample, typically a biological sample such as blood, saliva, or tissue. It involves breaking open the cells, removing proteins and other contaminants, and purifying the DNA so that it is free of other cellular components. The purified DNA can then be used for downstream applications such as PCR, sequencing, or cloning. Currently, it is a routine procedure in molecular biology or forensic analyses.

The Bradford protein assay was developed by Marion M. Bradford in 1976. It is a quick and accurate spectroscopic analytical procedure used to measure the concentration of protein in a solution. The reaction is dependent on the amino acid composition of the measured proteins.

<span class="mw-page-title-main">Triton X-100</span> Chemical compound

Triton X-100 is a nonionic surfactant that has a hydrophilic polyethylene oxide chain and an aromatic hydrocarbon lipophilic or hydrophobic group. The hydrocarbon group is a 4-(1,1,3,3-tetramethylbutyl)-phenyl group. Triton X-100 is closely related to IGEPAL CA-630, which might differ from it mainly in having slightly shorter ethylene oxide chains. As a result, Triton X-100 is slightly more hydrophilic than Igepal CA-630 thus these two detergents may not be considered functionally interchangeable for most applications.

<span class="mw-page-title-main">Plasmid preparation</span> Biological method of DNA extraction and purification

A plasmid preparation is a method of DNA extraction and purification for plasmid DNA. It is an important step in many molecular biology experiments and is essential for the successful use of plasmids in research and biotechnology. Many methods have been developed to purify plasmid DNA from bacteria. During the purification procedure, the plasmid DNA is often separated from contaminating proteins and genomic DNA.

Polysorbate 20 is a polysorbate-type nonionic surfactant formed by the ethoxylation of sorbitan monolaurate. Its stability and relative nontoxicity allows it to be used as a detergent and emulsifier in a number of domestic, scientific, and pharmacological applications. As the name implies, the ethoxylation process leaves the molecule with 20 repeat units of polyethylene glycol; in practice these are distributed across 4 different chains, leading to a commercial product containing a range of chemical species.

Alkaline lysis or alkaline extraction is a method used in molecular biology to isolate plasmid DNA from bacteria.

NUN buffer is a solution that makes it possible to purify proteins located in the nucleus of eukaryotic cells. Although other procedures are available they result in loss of albumin D-box binding protein (DBP) which is unwanted if nuclear signal pathways are to be investigated. Therefore, a new extraction procedure was developed in 1993 to increase recovery of nonhistone proteins using a (NUN) solution containing 0.3 M NaCl, 1 M urea, and 1% nonionic detergent Nonidet P-40, which destabilize salt bridges, hydrogen bonds, and hydrophobic interactions, respectively; resulting in a disruption of interaction between proteins and DNA. By incubating nuclei in NUN buffer and centrifuging the solution, the supernatant will therefore contain nuclear proteins.

<span class="mw-page-title-main">Proteinase K</span> Broad-spectrum serine protease

In molecular biology, Proteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Parengyodontium album. Proteinase K is able to digest hair (keratin), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 (subtilisin). The molecular weight of Proteinase K is 28,900 daltons.

<span class="mw-page-title-main">Spin column-based nucleic acid purification</span>

Spin column-based nucleic acid purification is a solid phase extraction method to quickly purify nucleic acids. This method relies on the fact that nucleic acid will bind to the solid phase of silica under certain conditions.

Radioimmunoprecipitation assay buffer is a lysis buffer used to lyse cells and tissue for the radio immunoprecipitation assay (RIPA). This buffer is more denaturing than NP-40 or Triton X-100 because it contains the ionic detergents SDS and sodium deoxycholate as active constituents and is particularly useful for disruption of nuclear membranes in the preparation of nuclear extracts. The stronger detergents in RIPA buffer cause greater protein denaturation and decrease protein-protein interactions.

Octyl glucoside is a nonionic surfactant frequently used to solubilise integral membrane proteins for studies in biochemistry. Structurally, it is a glycoside derived from glucose and octanol. Like Genapol X-100 and Triton X-100, it is a nonphysiological amphiphile that makes lipid bilayers less "stiff".

<span class="mw-page-title-main">Decellularization</span>

Decellularization is the process used in biomedical engineering to isolate the extracellular matrix (ECM) of a tissue from its inhabiting cells, leaving an ECM scaffold of the original tissue, which can be used in artificial organ and tissue regeneration. Organ and tissue transplantation treat a variety of medical problems, ranging from end organ failure to cosmetic surgery. One of the greatest limitations to organ transplantation derives from organ rejection caused by antibodies of the transplant recipient reacting to donor antigens on cell surfaces within the donor organ. Because of unfavorable immune responses, transplant patients suffer a lifetime taking immunosuppressing medication. Stephen F. Badylak pioneered the process of decellularization at the McGowan Institute for Regenerative Medicine at the University of Pittsburgh. This process creates a natural biomaterial to act as a scaffold for cell growth, differentiation and tissue development. By recellularizing an ECM scaffold with a patient’s own cells, the adverse immune response is eliminated. Nowadays, commercially available ECM scaffolds are available for a wide variety of tissue engineering. Using peracetic acid to decellularize ECM scaffolds have been found to be false and only disinfects the tissue.

References

  1. ↑ Posch, Anton (2014-12-01). "Sample preparation guidelines for two-dimensional electrophoresis". Archives of Physiology and Biochemistry. 120 (5): 192–197. doi:10.3109/13813455.2014.955031. ISSN   1744-4160. PMID   25211021. S2CID   42029377.
  2. ↑ Peach, Mandy; Marsh, Noelle; Miskiewicz, EwaI.; MacPhee, DanielJ. (2015-01-01). "Solubilization of Proteins: The Importance of Lysis Buffer Choice". In Kurien, Biji T.; Scofield, R. Hal (eds.). Western Blotting. Methods in Molecular Biology. Vol. 1312. Springer New York. pp. 49–60. doi:10.1007/978-1-4939-2694-7_8. ISBN   9781493926930. PMID   26043989.
  3. ↑ Posch, Anton (2008). 2D PAGE: Sample Preparation and Fractionation. Humana Press. pp.  24. ISBN   978-1-58829-722-8.
  4. 1 2 3 4 Affairs, EMBL - Office of Information and Public. "Protein Purification - Extraction and Clarification - Choice of lysis buffer and additives - EMBL". www.embl.de. Retrieved 2016-03-16.
  5. 1 2 Linke, Dirk (2009-01-01). "Chapter 34 Detergents: An Overview". In Richard R. Burgess; Murray P. Deutscher (eds.). Guide to Protein Purification, 2nd Edition. Methods in Enzymology. Vol. 463. pp. 603–617. doi:10.1016/s0076-6879(09)63034-2. ISBN   9780123745361. PMID   19892194.
  6. ↑ "Detergents for Cell Lysis and Protein Extraction". www.thermofisher.com. Retrieved 2016-03-16.
  7. ↑ Shehadul Islam, Mohammed; Aryasomayajula, Aditya; Selvaganapathy, Ponnambalam Ravi (March 2017). "A Review on Macroscale and Microscale Cell Lysis Methods". Micromachines. 8 (3): 83. doi: 10.3390/mi8030083 . ISSN   2072-666X. PMC   6190294 .
  8. ↑ Brown, Robert B; Audet, Julie (2008-10-06). "Current techniques for single-cell lysis". Journal of the Royal Society Interface. 5 (suppl_2): S131-8. doi:10.1098/rsif.2008.0009.focus. ISSN   1742-5689. PMC   2504493 . PMID   18426769.
  9. 1 2 3 Ji, Hong (2010-08-01). "Lysis of Cultured Cells for Immunoprecipitation". Cold Spring Harbor Protocols. 2010 (8): pdb.prot5466. doi:10.1101/pdb.prot5466. ISSN   1940-3402. PMID   20679375.
  10. 1 2 3 Sefton, Bartholomew M. (2001-01-01). "Labeling Cultured Cells with32Piand Preparing Cell Lysates for Immunoprecipitation". Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation. Vol. Chapter 18. John Wiley & Sons, Inc. pp. Unit 18.2. doi:10.1002/0471142727.mb1802s40. ISBN   9780471142720. PMID   18265167. S2CID   20969356.{{cite book}}: |journal= ignored (help)
  11. ↑ ACK Lysing Buffer
  12. ↑ "ACK Lysis Buffer". Cold Spring Harbor Protocols. 2014 (11): pdb.rec083295. 2014. doi:10.1101/pdb.rec083295.
  13. ↑ "A10492 - ACK Lysing Buffer - US".
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