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dsDNA is the double stranded DNA whereas ssDNA is the single stranded DNA, and although both of them carry genetic material they have a number of differences (Table 1).
|Abundance||Almost all organisms||Very few viruses (e.g. φX174)|
|Shape||Linear or filamentous||Stellate or star shaped|
|Stability||More stable||Less stable|
|A : T ratio||1||∼0.77|
|G : C ratio||1||1.3|
|Chargaff’s rule||Follows||Does not follow|
|Reaction to formaldehyde||Resistant||Highly susceptible|
|Purine : Pyrimidine ratio||1||Variable|
Although both DNA damage and DNA mutation are types of error in DNA, they are distinctly different from each other. DNA damages are physical abnormalities in DNA such as single- and double-strand breaks, while a mutation is a change in the base sequence of the DNA.
DNA damages can be recognized by enzyme and thus correctly repaired if redundant information is available for copying. While most DNA damages can undergo DNA repair, the un-repaired DNA damages can accumulate in replicating cells, giving rise to mutations.
In contrast to DNA damage, DNA mutations cannot be recognized and repaired by enzymes once the base change is present in both DNA strands, which can cause alterations in protein function and regulation.
DNA purity in a sample can be determined by measuring sample absorbance at 260 and 280 nm and determining their ratio, where the ratio of 1.7–2.0 indicates pure DNA sample. Contaminants which also absorb at 260 and 280 nm may cause overestimation of DNA in the sample.
While both optical density and absorbance both measure the absorption of light as it passes through a medium, optical density is related to the speed of light through the medium and takes refraction into account. On the other hand, absorbance does not take the refraction of light into account and only considers the amount of light lost.
Recombinant DNA (rDNA) are DNA molecules formed deliberately by laboratory methods (such as molecular cloning) to combine genetic materials from different sources, creating new sequences that would not otherwise be found in the genome. The difference between recombinant DNA and naturally occurring genetic recombination is that the former is created by artificial methods while the latter is a normal biological process existing in essentially all organisms.
The source of recombinant DNA can be from any species, such as plant, bacteria, human and fungal. If the DNA sequence does not occur in nature, it can even be created by the chemical synthesis of DNA. With recombinant DNA technology and synthetic DNA, literally any DNA sequence may be created and introduced into living organisms.
There are 3 major types of tight binding modes between DNA binding dyes and DNA:
Some dyes can bind to DNA’s major groove or externally bind with DNA through electrostatic interaction, though the binding is not as tight as the aforementioned three modes. Multiple binding modes can occur for one dye, and the dominant binding mode may change with different dye-DNA conditions such as dye-to-DNA ratio and DNA hybridization status.
Although both trypsin and pepsin are proteolytic enzymes secreted by the digestive system in order to digest proteins, they differ in many aspects.
An enzyme is a protein that acts as a catalyst to increase the biochemical reaction rate without altering itself in the process, while a coenzyme is an organic non-protein molecule that is required by an enzyme to perform its catalytic activity. Therefore, these two types of molecules differ in quite a few aspects:
HLA-DR and Iba1, which both serve as markers for human microglia, are not expressed evenly in all microglial tissue. HLA-DR, which is an MHC II cell surface receptor, serves mostly as a marker for active microglia. On the other hand, Iba1, or the ionized calcium binding molecule-1, is expressed in all microglia and plays a role in actin-binding and microglial membrane ruffling, rendering it better as a structural marker.
Primary cells or finite cells, are cells that are directly prepared from an organism’s tissue using enzymatic or mechanical methods. Under the right conditions, primary cells will grow and proliferate, however, they are only able to do so a finite number of times. Once that number is attained, cells enter a stage of senescence where they can no longer divide.
Cell lines are permanently established cell cultures that will proliferate indefinitely under the right conditions. Cell lines are preferably used for convenience as they are easier to handle and widely published, such as HeLa and HEK 293 cell lines.
Hoechst dyes are typically used for staining DNA content in live cells due to its high cell membrane permeability.
DAPI is typically used for staining DNA content in fixed cells due to its low membrane permeability.
The molar extinction coefficient (ε) for collagen is 52,940 cm-1M-1.
The molar extinction coefficient (ε) for Lysozyme is 38,940 cm-1M-1.
The molar extinction coefficient of RNA is:
The molar extinction coefficient (ε) for Ferritin is 8030 cm-1M-1.
The molar extinction coefficient (ε) for Rhodopsin at 500 nm (ε500) is 40,600 cm-1M-1.
The molar extinction coefficient (ε) for glucagon is 8,011 cm-1M-1.
The molar extinction coefficient (ε) for Insulin is 5,734 cm-1M-1.
The molar extinction coefficients for DNA are:
The extinction coefficients of DRAQ5 are:
Superoxide (O2–) itself has poor reactivity, but damages cells by promoting hydroxyl radical (.OH) formation, which in turn damages DNA in cells. The way this happens is that superoxide interacts with iron-sulfur clusters to obtain free iron to be used in the Haber-Weiss reaction, which generates hydroxyl radials from superoxide and hydrogen peroxide (H2O2).
Tumor protein p53 plays an important role in cell regulation and conserving the genomic stability, which is achieved by means of several mechanisms:
DNA (Deoxyribonucleic acid) extraction refers to the process of separating DNA from membranes, proteins as well as other materials in the cell that it is recovered from. DNA extraction can turn out to be the most exhaustive part in DNA analysis. Additionally, the methods of extraction might need an overnight incubation, might involve a recent procedure which deploys reagents and might be a procedure that may be completed in a couple of hours.
It is also worth noting that the process of DNA extraction involves careful handling of the biological material, thus preventing sample crossover and contamination. The used tubes during the process should be labelled carefully, particularly when transfer is needed.
mtDNA is the mitochondrial DNA that differs from nuclear DNA in a number of characteristics (Table 1).
|Abundance||Mitochondrial matrix||Nucleus of cell|
|Shape||Circular chain of DNA||Arranged in linear chromosomes|
|Number of copies||Multiple||Two|
|Number of genes||37||20,000 ? 30,000|
|Inheritance||Maternal||Maternal and paternal|
|Non-coding DNA genome proportion||3%||93%|
The HeLa cell line is classified as an immortal cell line because they can proliferate indefinitely, do not die after a set number of cell divisions and can be cultured for extensive periods of time. This cell line was originally derived from cervical cancer cells taken from Henrietta Lacks on February 8, 1951. They are the oldest and most frequently used cell line in scientific research.
The CHO cell line is an epithelial cell line derived from the ovary of Chinese hamsters. It has been used in biological and medical research since 1919, and in the commercial production of therapeutic recombinant proteins. CHO cells have found wide use in other notable research areas including genetics, gene expression and toxicity screening.
The differences between transfection and transformation are outlined in Table 2.
Table 2.The differences between transfection and transformation.
|Definition||Method of gene transfer in which the genetic material is deliberately introduced||Method of gene transfer in which the genetic material is directly uptaken and incorporated through the cell membrane(s)|
|Target cells||Mammalian cells||Plant, yeast and bacterial cells|
|Acheived by||Chemical, physical and viral methods||Chemical transformation, electroporation and particle bombardment|
Transfection is the process of deliberate introduction of naked or purified nucleic acids (DNA or RNA) into eukaryotic cells. Transfection can be achieved by viral and non-viral treatments. Viral mediated treatments include introduction of DNA by viral injection, using either retrovirus, lentivirus, adenovirus, adeno-associated virus, and herpes simplex virus. Non-viral treatments include physical (electroporation, cell squeezing, nanoparticles, magnetofection) and chemical treatments.
Both CRISPR interference (CRISPRi) and RNA interference (RNAi) are common techniques for gene silencing. The major difference between these two methods is that CRISPRi represses genes at the DNA level, whereas RNAi controls genes at the mRNA level. In other words, CRISPRi regulates gene expression primarily by inhibiting gene transcription, while RNAi destroys RNA transcripts. Comparing to RNAi, CRISPRi is associated with higher efficiency, greater versatility and fewer off-target effect.
Both EDTA and EGTA are chelating agents. They are aminopolycarboxylic acids that have more or less the same properties.
Ethylenediaminetetraacetic acid (EDTA) is a chelating agent consisting of six binding sites. It has the capacity to bind and sequester a variety of metal ions (except for alkali metals) such as Ca2+, Mg2+ and Fe2+. EDTA combines with all cations in a 1:1 ratio regardless of the charge on the cation. In laboratory applications, EDTA can be used as a preservative for biological samples. It scavenges for trace amounts of metal ions and prevents them from catalyzing air oxidation in the samples. EDTA has a higher affinity for Mg2+ ions compared to EGTA.
Ethylene glycol tetraacetic acid (EGTA) is also a chelating agent. Compared to EDTA, it has a higher affinity for calcium ions but a lower affinity for magnesium ions. Similar to EDTA, EGTA can be used as a buffer to resemble the pH of a living cell. This property of EGTA permits its usage in Tandem Affinity Purification, which is a protein purification technique. EGTA has a higher boiling point than EDTA.
Trypsin and chymotrypsin are two very similar digestive enzymes that hydrolyze proteins into amino acids. Although they share similar structure and function, there are still some differences between these two enzymes.
PBS and dPBS are the abbreviations of phosphate-buffered saline and Dulbecco’s phosphate-buffered saline, respectively. They are well-known buffer solutions that are commonly employed in biological research to maintain a consistent pH (between 7.2-7.6). The essential properties of both are that the ion concentrations and osmolarity retain their isotonic properties, meaning that the solutions are compatible with the human body. Although multiple formulations exist, the default for both will include sodium chloride (common table salt), and disodium hydrogen phosphate. Other ingredients, such as potassium chloride or potassium phosphate, may also be included in the formulation.
The substances can often be used interchangeably, although dPBS is typically slightly lower in phosphate concentration and may include calcium and/or magnesium. Experimental needs will dictate which solution should be employed.
For example, if in a particular experiment trypsin enzymatic activity will need to be measured, the calcium and magnesium sometimes included in dPBS might skew results, so simple PBS would be preferable.
Although both miRNA (micro RNA) and siRNA (small interfering RNA) are small RNA molecules involved in RNA interference and work through similar mechanisms, there are some differences between these two molecules.
The molar extinction coefficient (ε) for Immunoglobulin G (IgG) is ~210,000 cm-1M-1.
Primary cell culture is the culture of cells directly isolated from parental tissue of interest; whereas cell line is the culture of cells originated from a primary cell culture, which is generally used to expand cell population and prolong life span. These two processed differ in a few aspects.
Although there is a range for DNA purity and concentration, there are several factors that may impact a particular procedure, including the selected transfection reagent.
The DNA concentration can be determined by measuring the absorbance of the sample at 260nm in spectrophotometer and use the Beer-Lambert’s law to calculate the concentration. In order to do so, you would need to determine the extinction coefficient of the DNA given in cm-1M-1
Average Extinction Coefficient for bases in different nucleic acids:
ds DNA: Ec=6600 cm-1M-1, MW=330
ss DNA: Ec=8919 cm-1M-1, MW=330
RNA: Ec=8250 cm-1M-1, MW=340
Here is the way to calculate DNA Concentration:
dsDNA: 50 μg/mL O.D.=1; Con.(μg/mL)=Abs.x 50 μg/mL
ss DNA: 50 μg/mL O.D.=1.35; Con.(μg/mL)=(Abs./1.35)x 50 μg/mL
RNA: 50 μg/mL O.D.=1.21; Con.(μg/mlL=(Abs./1.21) x 50 μg/mL
DNA extraction is of main importance when it comes to studying genetic causes of various diseases and development of drugs and diagnostics. Additionally, it is important for conducting forensic science, detecting viruses and bacteria within the environment, detecting paternity and sequencing genomes.
There are 3 basic steps involved in DNA extraction, that is, lysis, precipitation and purification. In lysis, the nucleus and the cell are broken open, thus releasing DNA. This process involves mechanical disruption and uses enzymes and detergents like Proteinase K to dissolve the cellular proteins and free DNA.
The other step, which is known as precipitation, separates the freed DNA from the cellular debris. It involves use of sodium (Na+) ions to neutralize any negative charge in DNA molecules, making them less water soluble and more stable. Alcohol (e.g isopropanol or ethanol) is then added, causes precipitation of DNA from the aqueous solution since it does not dissolve in alcohol.
After separation of DNA from aqueous solution, it is then rinsed with alcohol, a process known as purification. Purification removes all the remaining cellular debris and unwanted material. Once the DNA is completely purified, it is usually dissolved in water again for convenient storage and handling.
The following is a sample protocol for the extraction of genomic DNA from cell culture.
Sample Size: Start with 1 x 105 to 5 x 106 cells.
DNA concentration can be determined either by absorbance or fluorescence. To determine DNA concentrations using the UV-absorbance method measure the absorbance of the DNA sample at 260 nm with a spectrophotometer.
Fluorescence methods determine DNA concentration by using double-stranded DNA binding dyes, such as Helixyte™ Green (AAT Bioquest Cat No. 17597) that fluoresce when bound to dsDNA, or DNA quantitation kits, such as the Portelite™ Fluorimetric High Sensitivity DNA Quantitation Kit (Cat No. 17661) or the Portelite™ Fluorimetric DNA Quantitation Kit with Broad Dynamic Range (Cat No. 17665). The fluorescence intensity is measured using a fluorometer, such as the CytoCite™ BG100 portable fluorometer from AAT Bioquest (Cat No. CBG100). Of the two methods, fluorescence-based DNA quantitation is more sensitive and generally used to quantify DNA for next generation sequencing.
Purity of DNA and RNA samples can be assessed by taking the ratio of absorbance at 260 nm and 280 nm (A260/A280). A ratio of ∼1.8 indicates a pure DNA sample, while a ratio of ∼2.0 indicates a pure RNA sample. If the ratio is higher or lower, it may indicate the presence of contaminates (e.g. protein, phenol, etc.) which also absorb at or near 280 nm. Strong absorbance around 230 nm can indicate that organic compounds or chaotropic salts are present in the purified DNA. A ratio of 260 nm to 230 nm can help evaluate the level of salt carryover in the purified DNA. The lower the ratio, the greater the amount of thiocyanate salt is present, for example. As a guideline, the A260/A230 is best if greater than 1.5.
|Reagent||Final Concentration||per 500 mL|
|1 M Tris pH 8.0||10 mM||5 mL|
|5 M NaCl||100 mM||10 mL|
|0.5 M EDTA pH 8.0||10 mM||10 mL|
|10% SDS||0.50%||25 mL|
|dH2O||to 500 mL|
DNA concentration in a sample can be determined in several ways:
Absorbance – DNA concentration is calculated by measuring the absorbance at 260 nm, and the turbidity at 320 nm, by using the following formula:
Concentration (µg/mL) = (A260 reading – A320 reading) × dilution factor × 50 µg/mL
Fluorescence – a method requiring a fluorescent DNA binding dye, a fluorometer that detects fluorescent dyes, and DNA standards to generate a standard curve. This method is more sensitive than the absorbance method and may be used for samples with low-concentration
Agarose Gel Electrophoresis – a method that requires horizontal gel electrophoresis tank, analytical-grade agarose, running buffer, intercalating DNA dye, and an appropriately sized DNA standard. The isolated DNA sample is loaded into a well of agarose gel alongside the standard. As the electric current flows, DNA migrates towards the anode, separating the DNA fragments by size as smaller DNA fragments travel faster than larger ones. The DNA in the gel can be visualized by an intercalating dye such as Cyber Green™ or Cyber Orange™ and then DNA can be quantified by comparing the sample with the standard.
No! Protein-coding DNA, which can be transcribed into mRNA that will be further translated into protein, makes up barely 2% of the human genome; more than 98% of DNA molecules are noncoding. Although non-coding DNA do not provide instructions for making proteins, they may encode for non-coding RNA such as microRNA, ribosomal RNA (rRNA), transfer RNA (tRNA), or enzymatic RNA molecules called ribozymes. In addition, non-coding DNA plays important roles in regulate cell functions, especially the control of gene activity.
The leading strand and lagging strand that are unwound by the DNA helicase at the replication fork run in opposite directions, but the new DNA sequences are synthesized in only one direction, i.e., 5’ to 3’ direction, because the enzymes involved in DNA replication can only work in the 5’ to 3’ direction. Therefore, these two template strands are replicated in different ways, resulting in the asymmetry of DNA replication.
During DNA replication, the leading strand can be continuously replicated by the polymerase since its template strand is in the 3’ to 5’ direction. However, the replication of the lagging stand is not so straightforward. It cannot be created in a continuous manner due to the 5’ to 3’ directionality of its template strand. Polymerases need to work backwards from the replication fork, creating periodic breaks in the process of replicating the lagging strand. DNA fragments, rather than continuous DNA sequence as in the leading strand, are generated in the lagging strand. These fragments, known as Okazaki fragments, are then connected into a single, continuous strand by the DNA ligase. In this way, the entire replication process is completed and it is considered asymmetric because of the difference in replicating these two strands.
DNA ligase a specific type of enzyme that joins DNA strands together by catalyzing the formation of phosphodiester bond. It can repair irregularities or breaks in the backbone of double stranded DNA molecules, hence playing an important role in DNA repair. Meanwhile, it is also used by cells in DNA replication, during which it connects Okazaki fragments on the lagging strand into a continuous strand. In addition, DNA ligase has become an indispensable tool in modern molecular biology research for generating recombinant DNA sequences. In molecular biology laboratories, purified DNA ligases are routinely used together with restriction enzymes to insert DNA fragments, often genes, into plasmids, creating a vector of interest.
While an excitation spectrum shows the wavelengths of light that a sample will absorb to be able to emit at a specified wavelength, an absorption spectrum shows all of the wavelengths at which light is absorbed by the sample. Generally, both the absorption and excitation spectra of a sample will peak at the same wavelength, although there are exceptions. In addition, absorption spectra is measured using a UV-Vis spectrophotometer, while excitation spectra is measured using a fluorescence spectrophotometer.
DNA cloning is a practice to create a large number of copies of a particular gene or other pieces of DNA. The cloned DNA can be used in many downstream applications, such as:
The concepts of EC50 and IC50, albeit similar, are not quite identical. While EC50 measures the concentration of a drug inducing its half-maximal effective response, IC50 represents the concentration of an inhibitor at which 50% of inhibition in its activity is achieved. A good way to remember the difference is using the acronym ‘I’ in IC50, which stands for inhibition, unlike ‘E’ in EC50, which refers to effective.
The molar extinction coefficient (ε) for Fluorescein is 70,000 cm-1M-1.
The term molar extinction coefficient (ε) is a measure of how strongly a chemical species or substance absorbs light at a particular wavelength. It is an intrinsic property of chemical species that is dependent upon their chemical composition and structure. The SI units of ε are m2/mol, but in practice they are usually taken as M-1cm-1. The molar extinction coefficient is frequently used in spectroscopy to measure the concentration of a chemical in solution.
You can use the Beer-Lambert Law to calculate a chemical species’ ε:
A = εLc
Rearrange the Beer-Lambert equation in order to solve for the molar extinction coefficient:
ε = A/Lc
Use the molar extinction coefficient to determine the brightness of a fluorescent molecule, by using the following equation:
Brightness = Extinction Coefficient (ε) x Fluorescence Quantum Yield (Φ)
An endosome is a membrane-bound compartment inside a eukaryotic cell. It is originated from the trans Golgi network and is an organelle of the endocytic membrane transport pathway, where various cargo molecules required for normal cellular function are internalized, recycled and modulated. Endosomes can be classified as early, sorting, or late depending on their stage post internalization, which mature into lysosomes at the end of the endocytic pathway. Their major function is to provide an environment for material to be sorted before it reaches the degradative lysosome.
DNA primase is a type of RNA polymerase. Since DNA polymerases can only recognize and elongate double-stranded sequences, the role of DNA primase in DNA replication is to catalyze and synthesize a short RNA segment (i.e., a primer) complementary to the ssDNA template, providing a double-stranded fragment for the DNA polymerase to recognize and thus initiating the replication. After elongation, the RNA piece will be removed by the 5’ to 3’ exonuclease and refilled with DNA. These DNA fragments are then joined together by DNA ligase, creating a single, continuous strand.
DNA repair is a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. Depending on the type of damage inflicted on the DNA molecule, a variety of repair mechanisms are adopted to restore the lost information, including direct reversal repair, single-strand repair, double-strand break repair, and translesion synthesis.
Exonucleases can act as proof-readers during DNA polymerization in DNA replication. They work by scanning along the newly synthesized strand directly behind the DNA polymerase. If the last nucleotide added is mismatched, it will be removed by the exonuclease. Therefore, exonucleases can be directly involved in repairing damaged DNA. Some polymerases (such as DNA polymerase I) have the intrinsic exonuclease activity derived from their exonuclease domains, which maintains the fidelity of DNA replication.
The main role of monovalent cations and ethanol is to eliminate the solvation shell that surrounds the DNA, thus allowing the DNA to precipitate in pellet form. Additionally, ethanol helps to promote DNA aggregation. Usually, about 70 percent of ethanol solution is used during the DNA washing steps. This allows the salts to dissolve while minimizing DNA solubility. The last 100 percent ethanol wash which is mainly employed helps to promote convenient ethanol evaporation from DNA pellet, thus preventing any carryover. Ethanol is preferred to water since its dielectric constant is lower.
The main reason is that RNA is less stable and easier to degrade compared to DNA. There are three main causes for RNA degradation:
Therefore, RNA isolation requires cautious handling of samples and good aseptic techniques. It is important to use only RNase-free solutions during the extraction, as well as RNase-free pipet tips and glassware.
The process to isolate mitochondria from cells involves two steps. The first step is cell disruption, which involves breaking open of the cell to release its cellular structure. It is generally referred to as cell lysis. There are different methods available to perform cell lysis. Physical techniques, like osmotic shock, simple freeze and grind, etc., can be used to physically break open the cell. Chemical methods like using detergents and biological lysis methods like using enzymes are also available to degrade the cell walls.
The second step is centrifugation. Mitochondria and other components of the broken cell can be separated by centrifugal force. All the broken cell components are rotated at different speeds. Depending on the size and density of the cell component, each component experiences different centrifugal force and deposits at the bottom. At a relatively high speed, mitochondria can be isolated form other cell components. A pellet of mitochondria will be deposited. Multiple rounds of centrifugation can be applied to get pure mitochondria.
Mitochondria are dynamic organelles capable of changing their organization and shape based on intracellular and extracellular signals. By balancing cycles of fusion and fission, mitochondria can regulate their morphology.
Mitochondrial fragmentation is the result of decreased fusion and increased fission in mitochondria. It is characterized by a large number of smaller mitochondria—as opposed to a network of highly interconnected and elongated mitochondria, which is the product of increased fusion.
Mitochondrial fragmentation is necessary for mitophagy, since smaller mitochondria are more easily engulfed by autophagosomes than larger ones and require less energy to be autophagocytosed. The fragmented state predominates during periods of high stress as well as before and after the release of apoptogenic factors, which signal for cell death.
Our MitoLite™ reagents are a set of fluorogenic probes for staining mitochondria of live cells. Any of our MitoLite™ reagents with “FX” in their name is a fixable mitochondrial stain, e.g., MitoLite™ Blue FX490 or MitoLite™ Red FX600.
MitoLite™ Green FM, similar to ThermoFisher’s MitoTracker Green FM (M7514), is one of the few mitochondrial reagents capable of staining mitochondria in dead cells. MitoLite™ Green FM can also stain mitochondria in live cells however; it is not well-retained after aldehyde fixation.
Properties for each MitoLite™ reagent is located in any of our MitoLite™ protocols see link in Additional Resources below.
Rough ER is involved in protein storage and synthesis, and tends have higher density around the nucleus. It has ribosomes bound to the cytosolic side of the membrane which are responsible for translation. Proteins are also folded in the lumen of rough ER.
Smooth ER is mainly involved in lipid metabolism and steroid production, as well as lipid storage. In muscle cells, smooth ER also plays the critical role of storing and releasing calcium ions. Unlike rough ER, smooth ER does not have ribosomes bound to its surface.
Stable and transient transfection differ in their long-term effects on a cell. In stable transfection, the plasmid DNA successfully integrates into the cellular genome and will be passed on to future generations of the cell. However, in transient transfection, the transfected material enters the cell but does not get integrated into the cellular genome. Thus, a transiently transfected cell will only express transfected DNA for a short amount of time and not pass it on to daughter cells.
Transfection reagent is the chemical compound used to achieve transfection (i.e. introduce nucleic acid into eukaryotic cells). Transfection reagents, such as Transfectamine™ 5000, are positively charged and attract the negatively charged DNA to form a positively charged polymer, which can interact with negatively charged cell membrane which enables the uptake of this polymer into the cell. The polymer then travels to the cell nucleus and releases (transfects) the DNA.
Transfection is the process of introducing nucleic acids into cells. With transduction, nucleic acids are introduced into the cell specifically by means of a viral vector. The expression of genetic material from these two methods can either be transient, where the cell does not incorporate the foreign gene into its genome, or stable, where it is incorporated into the genome. Stable transfection/transduction can also be called permanent transfection/transduction.
If you are developing an in-house procedure or making use of a new transfection reagent, we suggest these initial preparation steps:
Transfection procedures and reagents have improved over recent years, but some degree of cell damage and labeling inefficiency persists. If your transfection protocols are giving disappointing results, some possible causes include:
Your optimal protocol will depend on multiple factors including your chosen cell line, but we do have a suggested protocol for our transfection reagent.
Sample Protocol below or available here:
1. Prepare cells for transfection
2. Prepare Transfectamine™ 5000-DNA mixture
3. Add Transfectamine™ 5000-DNA mixture to cell culture
4. Culture overnight
5. Analyze transfection efficiency with appropriate method
There are multiple ways to improve your transfection results with a particular cell line and reagent.
We suggest the following:
Liposome transfection is a technique of inserting genetic material into cells using liposomes. In liposome transfection, cationic lipids are used to form liposomes, which take up nucleic acids. These nucleic acids can be DNA or siRNA. The positive charge of the liposomes and negative charge of the nucleic acids allow the two to form a complex, which can then enter the cell through endocytosis.
Transfection efficiency is the percentage of cells that are transfected compared to the entire population. It usually can be evaluated by measuring the expression of reporter genes on the transfection plasmids, such as GFP.
The general steps of ion-exchange chromatography procedure are as follows:
CRISPR interference (CRISPRi) is a genetic perturbation technique that inhibits gene expression by targeting a nuclease dead version of Cas9 (dCas9) to a region near the transcription start site (TSS). Thereby, the cell’s transcription machinery is prevented from accessing the TSS, resulting in the inhibition of gene expression.
CRISPR activation (CRISPRa) is also a type of CRISPR tool that employs the modified versions of dCas9. In contrast to CRISPRi, transcriptional activators are added on dCas9 or the guide RNAs, aiming to increase, rather than inhibit, expression of genes of interest.
RNA extraction is the purification of RNA from biological samples. Several methods are used in molecular biology to isolate RNA from samples, including guanidinium thiocyanate-phenol-chloroform extraction, glass fiber filters based on silica technology, magnetic beads assisted purification, as well as column chromatography. Among these methods, guanidinium thiocyanate-phenol-chloroform extraction, using commercially available TRIzol (TRI reagent), is the most common one, which isolates RNA from DNA and proteins based on their different solubilities in aqueous and organic solutions.
RNA splicing is a form of RNA processing for the maturation of mRNA. During splicing, introns in the precursor messenger RNA (pre-mRNA) are removed and exons are joined, leading to the formation of a mature messenger RNA (mRNA). Several methods of RNA splicing exist in nature, for example, self-splicing, tRNA splicing and splicing by spliceosome. The type of splicing depends on the organism, structure of intron, and the requirement of catalysts.
RNA molecules in the cytosol clump together into ‘granules’ for easy transportation by the protein annexin A11. This protein, once it has attached to an RNA granule, will adhere to lysosomes, which travel easily throughout the cell. This assisted travel is essential in larger cells such as neurons. Once the RNA has been delivered to the correct location within the cell, its code can be translated into the necessary protein. Dysregulation of RNA transportation means that proteins either are not manufactured in the first place, or are in the wrong locations, so they cannot be used effectively. Multiple pathologies, such as ALS, are linked to errors in this transportation mechanism, often with the protein annexin A11 or some other aspect of the RNA transportation system. Tracking lysosome activity is useful in both living and fixed cells to measure multiple cell processes, including RNA translation.
For more information on RNA transportation in large cells, see the full paper in Cell.
Metabolism, which is the set of all biochemical reactions in a cell or body, is comprised of both anabolism and catabolism.
Anabolism comprises of constructive reactions involved in synthesizing complex molecules. Anabolic reactions are endergonic and require energy to occur. On the other hand, catabolism includes all the destructive reactions involved in the breaking down of complex molecules. Catabolic reactions are exergonic and release energy.
PBS is often used for washing due to being isotonic and non-toxic to cells and tissues, and thus allows for cells to be rinsed of unwanted media without potentially lysing them.
The siRNA is a short double-stranded RNA that is derived from foreign RNA molecules uptaken by cells. During RNAi, the ribonuclease protein “Dicer” is first activated, cutting these exogenous long dsRNA into small fragments of 20-25 base pairs, i.e., siRNA. Then, these siRNAs integrate into a multi-subunit protein called the RNA-induced silencing complex (RISC) and are separated into single strands. One of the two single strands is degraded, while the remaining one is available to base-pair to its target mRNA. Once mRNA is bound to siRNA, mRNA will be cleaved and destroyed. However, the siRNAs remain unharmed throughout the process, which can bind to and destroy other newly-synthesized matching mRNA molecules. In this way, no mRNA is available for translation; thus, protein production from the target gene is silenced.
Cell culture can be contaminated in a number of ways, broadly classified as chemical contamination (endotoxins, free radicals, heavy metals, plasticizers, detergents or disinfectants) and biological contamination (Bacteria, Yeast, Mold, Viruses, Mycoplasmas, Protozoa, Invertebrates and cross-contamination from other cell lines). The contamination can be detected by visual inspection (e.g. detecting cloudiness in the media); microscopic examination (which can detect bacteria and viruses); testing pH (as contaminants may increase or decrease the pH).
There are four main phases in the growth curve of normal cultured cells, which typically displays a sigmoid pattern of proliferation.
The purpose of CO2 in cell culture is to maintain a stable physiological pH through the CO2-bicarbonate based buffer system. The atmospheric CO2 can dissolve into cell culture medium, and a small portion of it will react with water to form carbonic acid, which in turn interacts with the bicarbonate ion. The balance of dissolved CO2 and bicarbonate thereby controls the pH of the medium. For most cell culture experiments, 4-10% CO2 in air is commonly used.
Serum has been widely used as a supplement for the in vitro cell culture. It is an important source for growth and adhesion factors, hormones, lipids and mineral. Besides, it participates in the regulation of cell membrane permeability, and can serve as a carrier for lipids, enzymes, micronutrients, and trace elements into the cell.
β-Galactosidase is an intracellular enzyme that is an essential part of the cellular metabolism of galactosides like lactose. It cleaves (separates) large substrate molecules into smaller ones by breaking the glycosidic bond. This enzyme is essential for energy production in most forms of multicellular life.
Yes. Antibiotics can be used in cell cultures to prevent bacterial infection. However, antibiotics should never be used routinely, because they can impair cell growth and differentiation. Besides, the continuous use of antibiotics can encourage the development of antibiotic-resistant strains and allow low-level contamination to persist. Once the antibiotic is removed from the media, a full-scale contamination may be developed. Therefore, antibiotics should only be used as a last resort and only for short-term applications, which should be removed from the culture as soon as possible.
Monoclonal antibodies bind to one unique epitope on an antigen, while polyclonal antibodies bind to more than one type of epitope on an antigen. This occurs because monoclonal antibodies are produced by the same clone of plasma B cells, making the antibody population homogenous. Polyclonal antibodies have a heterogenous antibody population since they are produced by different clones of plasma B cells. While monoclonal antibodies tend to have lower cross-reactivity and produce lower background than polyclonal antibodies, the higher overall affinity and sensitivity of polyclonal antibodies can be beneficial when detecting low quantities of protein.
Anti-human antibodies are effective for detecting human antibodies, while anti-mouse antibodies are effective for detecting mouse antibodies. While different antibodies may be reactive towards the same type of antigen (eg. anti-human CD45 and anti-mouse CD45 antibodies), they are most specific for the species that they were raised against.
Antibody labeling, or antibody conjugation, is the process of covalently attaching a label (e.g. an enzyme or fluorophore) to a primary or secondary antibody. These labels have the capacity to generate a measurable signal that facilitates in the detection of the antibody-antigen complex. Antibody conjugates are widely used in a broad range of immunological applications including Western blot, ELISA, flow cytometry, immunohistochemistry (IHC), immunocytochemistry (ICC) and immunofluorescence (IF).
Two methods are commonly used to label antibodies. The first, and the simplest method, is to label primary amines (-NH2) that exist at the N-terminus of each polypeptide chain and in the side-chain of lysine residues of antibodies. This requires the use of fluorophores modified with amine-reactive chemical groups such as succinimidyl esters (SE) and NHS esters. The second method is to label thiol groups (-SH) that are located in the side-chain of cysteine residues. This requires the use of fluorophores modified with thiol-reactive chemical groups such as maleimides.
If conjugation chemistry is not your strong suit, consider using antibody labeling kits such as ReadiLink™ Rapid iFluor™ Dye Antibody Labeling Kits. These kits produce fluorescent antibody conjugates in two easy mixing steps with 100% conjugate recovery.
Choosing appropriate primary and secondary antibodies for western blotting is much like choosing antibodies for other types of assays—the primary antibody should be specific to the protein of interest and should be of a different host species than the sample, while the secondary antibody should be reactive against the host of the primary antibody. In addition, the primary antibody should be validated for use in western blotting, and should be specific for either the denatured or native conformation of the protein of interest, depending on the type of PAGE (SDS or native) performed.
Primary and secondary antibodies should not be incubated at the same time. Incubating primary and secondary antibodies together has the potential to form a large complex of antibodies which never binds to the antigen on the membrane. Primary and secondary antibodies also require different incubation times. In addition, not applying the primary and secondary antibodies sequentially also makes it more difficult to analyze poor results, since it would hard to discern whether the result is due to the primary, the secondary, or their interaction.
To dilute from a ratio of 1:200 to 1:1000, you should perform a 5 times dilution. Divide the desired total volume of the new solution by the dilution factor (in this case, 5) to obtain the volume of initial antibody solution that should be used. The total desired volume minus this number is the volume of dilution buffer that should be added.
For example, if 1 ml of 1:1000 solution is desired, you would add 200 ul of 1:200 antibody solution to 800 ul of dilution buffer.
SDS-PAGE is an electrophoresis method that separates proteins by mass. Western blot is an analytical technique to identify the presence of a specific protein within a complex mixture of proteins, where gel electrophoresis is usually used as the first step in procedure to separate the protein of interest. SDS-PAGE is by far the most common type of gel electrophoresis being used in western blot.
Enhanced chemiluminescence (ECL) is a detection technique based on the chemiluminescence of substrates such as luminol and acridan. Due to its high sensitivity, wide dynamic range, and high signal-to-noise ratio, ECL is one of the most popular detection methods for a variety of western blotting applications, and is also widely used for quantifying biological analytes such as DNA, RNA as well as cells.
In a typical ECL assay, antibodies that specifically recognize the molecule of interest are first labeled with horseradish peroxidase (HRP). A chemiluminescent substrate and an oxidizing agent (hydrogen peroxide) are then catalyzed by HRP to produce excited intermediates, which release a strong blue emission at 450 nm wavelength upon decaying to the ground state. The light emissions can be captured with an x-ray film and/or detected by a luminescent signal instrument.
The term “enhanced” is derived from the enhancer being used together with the chemiluminescent substrates. Without an enhancer, the light emitted is usually of low intensity and decays too fast to make an accurate detection and analysis. With an enhancer (e.g. modified phenol, naphthol, aromatic amine or benzothiazole), the reactions can proceed for prolonged duration (up to several minutes) without significant reduction in light output, allowing for accurate and sensitive detections.
Sometimes referred to as ‘crossover’, this common microscopy problem refers to overlapping excitation and emission wavelengths of two or more fluorescent dyes, which muddy the signal and interfere with accurate measurement of experimental results. Overlapping spectra can give false negatives or positives, or otherwise obscure data.
To prevent this issue for multiparameter visualizations, dyes with good separation between their excitation and emission spectra should be chosen. Some dyes have wider spectra bands than others, so the researcher must take this into account. If one or more dyes must be used that will potentially overlap, choosing multiple controls (negative controls, single-dye, and others) will help compensate for the issue during data analysis. Another method of compensation is to use more narrow bandpass filters, which will help sanitize the signals, but at the cost of lower overall signal levels.
Careful dye selection and instrumentation, along with the use of experimental controls, will minimize the presence of fluorescence crosstalk.
AAT Bioquest’s Interactive Spectrum Viewer allows easy visual comparison of the presence and degree of spectral overlap between hundreds of commonly selected fluorophores.
All three techniques are similar in that they use antibodies to selectively identify targets of interest in cells or biological tissue sections, either directly or indirectly. In direct detection methods, a single primary antibody is conjugated to a detectable tag, such as an enzyme or fluorophore, and is used in a single-step procedure to directly detect the target of interest. With indirect detection, two antibodies are used in sequence for the detection of a target antigen. First, the sample is incubated with an unlabeled primary antibody directed against the target antigen. Then, a labeled secondary antibody specific for the primary antibody is used to detect its presence, and thus the target of interest.
Generally speaking, for antibodies conjugated to an enzyme (e.g. HRP) visualizing the antibody-antigen interaction requires a substrate specific to the enzyme tag being used. The enzyme catalyzes a color-producing reaction with its respective substrate, and the resulting chromogenic signal can then be detected using either a spectrophotometer or an absorbance microplate reader. For antibodies conjugated to a fluorophore, detect using a fluorescence instrument (e.g. fluorescence microscope, fluorescence microplate reader or flow cytometer).
The three staining techniques differ in the sample/tissue type:
Bacteria and viruses differ in many ways, such as structure, size, pathogenicity as well as response to medications.
The aldehyde functional group of glutaraldehyde allows it to act as a preservative by binding to amine groups and ultimately keeping proteins in a cell intact. However, this process causes autofluorescence due to the binding of glutaraldehyde to labeled antibodies. In order to quench this fluorescence, the aldehyde groups must be reduced so that the binding does not occur. To do this, a reducing agent such as sodium borohydride or Schiff’s reagent can be used.
Exon shuffling is a molecular mechanism for the formation of new genes, where two or more exons from different genes are recombined between introns, yielding rearranged genes with altered functions. There are different mechanisms for exon shuffling, such as transposon mediated exon shuffling, crossover during sexual recombination of parental genomes and illegitimate recombination.
An enzyme inhibitor is a molecule that binds to an enzyme and decrease its activity, thus decreasing the reaction rate. The binding of an inhibitor can stop a substrate from entering the active site of the enzyme, hindering the enzyme from catalyzing the reaction. Consequently, the amount of product produced by the reaction is decreased, which is inversely proportional to the concentration of inhibitor molecules.
Inhibitors are classified into two categories, reversible and irreversible inhibitors, based on the nature of binding with enzyme.
Cell division, as part of a cell cycle, is the process by which a parent cell divides into two or more daughter cells. In eukaryotes, there are two types of cell division: mitosis and meiosis. During mitosis, a cell duplicates all its contents, including its chromosomes, and splits to form two identical daughter cells. Meiosis, on the other hand, reduces the chromosome number by half. It is the type of cell division that creates egg and sperm cells, which ensures that humans have the same number of chromosomes in each generation.
Restriction enzymes are generally categorized into four groups, types I, II,III and IV, which differ primarily in structure, cofactor, cleavage site and specificity.
There are at least four distinct types of introns:
There are 4 types of reversible enzyme inhibitors.
Ion-exchange resins are a network of hydrocarbons formed by organic polymers, to which charged functional groups, who are the ion exchange sites, are affixed. These functional groups readily attract biomolecules of the opposite charge.
There are two main types of ion-exchange resins: cation exchange resins and anion exchange resins. Cation exchange resins are negatively charged which are used for separating cation analytes. Anion exchange resins, on the other hand, are positively charged for anion analytes.
Ion-exchange resins are also categorized as “strong” or “weak” exchangers. The categorization is not related to the strength of ion binding, but based on the extent that the ionization state of the functional groups varies with pH. Strong exchangers, such as quaternary ammonium (Q) and sulfopropyl (SP), remain fully charged over a broad range of pH, showing no variation in ion exchange capacity, which makes optimization of separation simpler. Weak exchangers, like diethylaminoethyl (DEAE) and carboxymethyl (CM) can only be ionized over a limited pH range. Weak exchangers usually have better selectivity than the strong ones because of this added variation in ionization.
Proteases catalyze the proteolysis through different mechanisms, which can be classified into 6 main groups based on the different active site residues they employ to perform catalysis.
Chromatography is a separation technique used to isolate the individual components in a mixture, in which a mobile phase carries the mixture travelling through a stationary phase at different speeds. The differential partitioning between the mobile and stationary phases, which is referred to as a compound’s partition coefficient, results in differential retention on the stationary phase, causing them to separate.
Chromatography is categories as column chromatography and planar chromatography based on their different bed shape.
Column chromatography, with its stationary bed contained in a tube, can be further divided into gas chromatography and liquid chromatography based on the physical state of mobile phase. Liquid chromatography is now being widely used in biochemistry, pharmaceuticals and food analysis as a standard separation and purification method.
Planar chromatography, on the other hand, presents its stationary phase on a plane, such as a paper (paper chromatography) or a glass plate (thin-layer chromatography). It is usually cheaper, less sophisticated, and easier to manipulate than the column chromatography, which is constantly used in labs for fast screening of a series of compounds.
IC50 of a drug is obtained by generating a dose-response curve and analyzing the drug-inhibitor interaction at different concentrations. Based on the plot of the dose-response curve, IC50 values are derived for specific inhibitors by determining the concentration required to reduce 50% of the maximum response from the drug. Generally, for a fixed concentration of inhibitory substance, a higher inhibition indicates a lower drug response and hence, a smaller IC50 value. For a quick solution, use our free online IC50 calculator, Quest Graph™ IC50 Calculator, which allows for an accurate and efficient approach to determining IC50 values representative of your data set.
IC50 represents the concentration at which a substance exerts half of its maximal inhibitory effect. This value is typically used to characterize the effectiveness of an antagonist in inhibiting a specific biological or biochemical process (ex. phosphorylation).
In pharmacology, it is an important measure of potency for a given agent. As reported by the FDA, the IC50 value represents the minimal concentration of a drug that is required for 50% inhibition in vitro. Traditionally, this value is expressed as a molar concentration.
The difference between these two chromatography methods is derived from their different working principles. Ion-exchange chromatography is used to separate charged analytes, which is based on the electronic interaction between the column and the target molecule who has an opposite charge to that of the stationary phase surface. However, for affinity chromatography, it proceeds because target molecules, whether charged or not, have a high affinity for the stationary phase due to some specific interactions such as antigen-antibody interactions and enzyme-substrate interactions.
Bacteria prevent cutting their own DNA by masking the restriction sites with methyl groups (CH3). The methylation process is achieved by the modification enzyme called methyltransferase. Bacterial DNA is highly methylated and is unrecognizable for the restriction enzymes, thus being prevented from cleavage.
LacZ is a frequently used reporter gene, encoding for the protein beta-galactosidase in cultured cells, which appear blue when the cultured cells are grown on a medium containing X-gal analog.
The steps for lacZ staining are:
SDS (sodium dodecyl sulfate) is an anionic detergent that unfolds and denatures proteins, coating proteins in negative charge. It is added in excess to the proteins, so that the proteins’ intrinsic charge is covered, and a similar charge-to-mass ratio is obtained for all proteins. In this way, the migration rate of proteins will be dependent on their size, but not their intrinsic charge.
The E. coli LacZ gene is often used as a reporter gene since it produces a blue product once it is cleaved by the β-galactosidase enzyme. This ‘reports’ whether or not the gene is expressed by the bacteria when grown in a compatible substrate (such as X-gal).
Magnesium is essential for many cellular pathways. First, magnesium is a crucial activator of ATP, and acts as a cofactor for many essential enzymes that require ATP to function. These enzymes include ATPases that are involved in ion transport, as well as protein kinases, which function to activate other enzymes by phosphorylation.
Magnesium also promotes the activity of numerous enzymes such as mitochondrial dehydrogenases, one of which is 2-oxoglutarate dehydrogenase (OGDH), a rate-limiting enzyme for the citric acid cycle. It is also important for the activity of isocitrate dehydrogenase (IDH) and pyruvate dehydrogenase complex (PDH).
In addition, magnesium is also important for regulating ion channels, especially voltage-dependent Ca2+ channels and K+ channels.
Cell proliferation can be determined by measuring such parameters as newly synthesized DNA, total nucleic acid content or cell division (Table 1).
The simplest method would be to monitor cell division using amine-reactive cell tracking indicators, such as CytoTell™ dyes. These cell-permeable indicators covalently bind to cytoplasmic proteins. Due to this covalent coupling reaction, CytoTell™ dyes cannot be transferred to adjacent cells and are well-retained in cells for several generations (up to 9 generation can be visualized). As cells divide, CytoTell™ dyes are distributed equally between daughters cells, and each new generation of cells is marked by a fluorescence intensity half that of its parents. Cells labeled with CytoTell™ dyes may be fixed and permeabilized using standard formaldehyde-containing fixatives and saponin-based permeabilization buffers for further intracellular analysis.
Table 1. List of Reagents and Assays for Monitoring Cell Proliferation.
|Monitor Cell Division||Uses cell-permeable dyes that bind to cytoplasmic proteins. As cells divide, dye is transferred to daughter cells. Each new generation of cells is marked by fluorescence intensity half its parents.||CytoTell™ dyes||Fluorescence microplate reader, fluorescence microscope or flow cytometer|
|Monitor Newly Synthesized DNA||Incorporates modified nucleotides into newly synthesized DNA during the S-phase of the cell cycle. Once incorporated, these nucleoside analogs serve as cell cycle and proliferation markers that can be detected using labeled probes to identify cells that are actively proliferating.||Bucculite™ dT Incorporation Cell Proliferation Fluorescence Imaging Kits||Fluorescence microscope|
|BrdU (requires an enzyme or fluorophore labeled anti-BrdU antibody)||Can be adapted for colorimetric, fluorimetric or chemiluminescent instruments.|
|Monitor Total Nucleic Acid Content||Uses cell-permeable nucleic acid stains that exhibit emission signals proportional to DNA mass. Flow cytometric analysis of stained populations is then used to generate a DNA histogram to reveal the percentage of cells in each phase of the cell cycle.||Cell Meter™ Fluorimetric Live Cell Cycle Assays||Flow cytometer|
|Cell Meter™ Fluorimetric Fixed Cell Cycle Assays|
|Nuclear Violet™ LCS 1|
|Hoechst 33258, Hoechst 33342 and DAPI|
M1 macrophages are seen to be involved in pro-inflammatory and immune responses. CD80, CD86, iNOS, and MHC-II are all markers that can be used to identify M1 macrophages.
Meanwhile, M2 macrophages are involved with cell proliferation and tissue repair, and can be identified by cell surface markers CD206, CD209, and CD163.