AAT Bioquest FAQs

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.

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:

1. Intercalating (e.g. Propidium iodide)
2. Minor-groove binding (e.g. DAPI)
3. Bis-intercalating (e.g. YOYO-1)

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.

• Origin: Pepsin is the chief digestive enzyme in stomach, which is produced by the gastric gland in stomach and is a component of gastric juice, while trypsin in produced by the pancreas and is a component of pancreatic juice.
• Activation: The inactive form of pepsin, pepsinogen, is activated by HCl of the gastric juice, whilst the inactive form of trypsin, trypsinogen, is activated by an enzyme called enterokinase.
• Catalysis mechanism: Pepsin is an aspartic protease which uses a catalytic aspartate in its active site, while trypsin is a serine protease employing the serine residue in active site.
• Optimal pH: The optimum pH for pepsin activity is 1.8, while trypsin works best in alkaline pH (pH 7.5-8).
• Types: Pepsin has four different types, i.e. pepsin A, B, C and D, while trypsin has two types, ?- and ?-trypsin.
• Specificity: Pepsin hydrolyzes peptide bonds between large hydrophobic amino acid residues, whereas trypsin hydrolyzes peptide bonds at the C-terminal side of lysine or arginine.
• Function: Pepsin acts on proteins and converts them into peptones, while trypsin converts peptones into polypeptides.

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:

• Size: Enzymes are large molecules, while coenzymes are usually small molecules.
• Nature: Enzymes are mainly globular proteins, whereas coenzymes are non-protein molecules.
• Function: Enzymes are biological catalysts; while coenzymes are helper molecules to the enzymes, which is necessary for the enzyme to execute its catalytic activity.
• Change of structure: Enzymes’ structure remains unaltered throughout the reaction, whereas coenzymes are chemically changed after the enzymatic reaction.

Primary Cells versus Cell Lines

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.

52940 cm^-1M^-1

The molar extinction coefficient (ε) for collagen is 52,940 cm^-1M^-1.

38,940 cm^-1M^-1

The molar extinction coefficient (ε) for Lysozyme is 38,940 cm^-1M^-1.

The molar extinction coefficient of RNA is:

• 40 (μg/mL)⁻¹cm⁻¹ (Absrobance max at 260 nm)

8030 cm^-1M^-1

The molar extinction coefficient (ε) for Ferritin is 8030 cm^-1M^-1.

40,600  cm^-1M^-1

The molar extinction coefficient (ε) for Rhodopsin at 500 nm (ε₅₀₀) is 40,600 cm^-1M^-1.

8011 cm^-1M^-1

The molar extinction coefficient (ε) for glucagon is 8,011 cm^-1M^-1.

5734 cm^-1M^-1

The molar extinction coefficient (ε) for Insulin is 5,734 cm^-1M^-1.

The extinction coefficients of DRAQ5 are:

• 22,000 cm^-1M^-1 in Methanol (Absorbance max at 647 nm)
• 12,000 cm^-1,M^-1 in PBS (Absorbance max at 600 nm).

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:

• Activating DNA repair proteins when DNA has sustained damage.
• Arresting growth by holding the cell cycle at the G1/S regulation point on DNA damage recognition.
• Initiating apoptosis if DNA damage is irreparable.
• Serving as a critical component for the senescence response to short telomeres.

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.

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.

Both EDTA and EGTA are chelating agents. They are aminopolycarboxylic acids that have more or less the same properties.

EDTA

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.

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.

• Specificity: Trypsin hydrolyzes peptide bond at the C-terminal side of basic amino acids such as lysine and arginine, whereas chymotrypsin attacks the C-terminal side of aromatic amino acids like phenylalanine, tryptophan, and tyrosine. This is the main difference between these two enzymes.
• Activation: The inactive form of trypsin, trypsinogen, is activated by enterokinase, while chymotrypsinogen is activated by trypsin.

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.

• Origin: The siRNA is an exogenous double-stranded RNA uptaken by cells, while miRNA is single-stranded and comes from endogenous non-coding RNA. Besides, the siRNA is present in lower animals and plants, but not found in mammals; whereas miRNAs are present in all the animal and plant.
• Structure: The siRNA is a 21-23 nucleotide long RNA duplex with a dinucleotide 3’ overhang, whereas the miRNA is a 19-25 nucleotide RNA hairpin which forms duplex by binding with each other.
• Target: The siRNA is highly specific with only one mRNA target, while miRNA can inhibit translation of multiple mRNA targets because of its imperfection in pairing.
• Purpose: The siRNA is primarily to provide viral defense and genome stability while the miRNA functions as endogenous gene expression regulator.

~210,000 cm^-1M^-1

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.

• Resemblance to parental tissue: Cells in primary culture closely resemble the parental tissue, while cells in a cell line might have mutations or genetic alterations during sub-culturing.
• Process of obtaining cells: In primary cell culture, cells are isolated from tissues, which usually go through phases of rinsing, dissection, mechanical or enzymatic disaggregation, and separation. In contrast, obtaining cells for a cell line is much more straightforward, which are directly transferred from the primary cell culture. If the primary cell is an adherent type, a detaching step is required.
• Life span: Primary cell cultures have finite life spans because the growth of cells exhausts substrate and nutrients, during which toxic metabolites are also accumulated, leading to the death of cells. However, cell lines have prolonged lifespan. Periodic sub-culturing can even produce immortal cells through transformation or genetic alteration of primary cells.
• Risk of contamination: Primary cell cultures are more difficult to take care of, which has a higher risk of contamination than the cell line.

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.

We suggest:

• Compare DNA/Reagent concentrations for best performance

• Test different incubation periods of DNA and reagent before cell mixing

• Endotoxin levels should be kept as low as possible

• A₂₆₀/A₂₈₀of 1.7-1.9 are ideal purity levels for most procedures-try in this range first and adapt from there

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 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.

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 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:

• Working out the function of the gene
• Investigating a gene’s characteristics (size, expression, tissue distribution)
• Studying how mutations may affect a gene’s function
• Production of protein coded for by the gene

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

Where:

• A is the amount of light absorbed by the sample for a particular wavelength
• ε is the molar extinction coefficient
• L is the distance that the light travels through the solution
• c is the concentration of the absorbing species per unit volume

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)

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.

• Direct reversal repair: It is a mechanism of repair where specialized proteins eliminate the DNA damage by chemically reversing it. It is the simplest and most energy efficient form of DNA repair, which does not require a refence template. Moreover, it does not involve the process of breaking the phosphodiester backbone of the DNA.
• Single-strand repair: When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. Three types of mechanisms are commonly employed to repair the single-strand damage, including Base excision repair (BER), Nucleotide excision repair (NER) and Mismatch repair.
• Double-strand break repair: When both strands in the double helix are severed, DNA molecule forms double-strand breaks. Three mechanism exist to repair these double-strand breaks: non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homologous recombination (HR).
• Translesion synthesis: When cells have no access to a template to recover the original information, they use translesion synthesis as a last resort, which allows the DNA replication machinery to replicate past DNA lesions such as thymine dimers or AP sites.

The main reason is that RNA is less stable and easier to degrade compared to DNA. There are three main causes for RNA degradation:

• RNA is more reactive than DNA because of the ribose units in its structure, which have a highly reactive hydroxyl group on C2 that takes part in RNA-mediated enzymatic events.
• RNA is single-stranded, while DNA is mostly double-stranded. RNA has larger grooves than DNA, which makes it easier to be attacked by enzymes.
• Enzymes that degrade RNA, ribonucleases (RNases) are abundant in environment and hard to be removed completely. For example, autoclaving a solution containing bacteria will destroy the bacterial cells but not the RNases released from the cells. Furthermore, even trace amounts of RNases are able to degrade RNA.

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.

If you are developing an in-house procedure or making use of a new transfection reagent, we suggest these initial preparation steps:

• An initial cell viability assay to ascertain cell health

• 70-90% confluency recommended for most cell lines

• Minimize required reagent to keep toxicity low

• Prepare cell culture 24 hours prior to procedure for optimal replication state

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:

• Reagent may not have been stored correctly. Check to ensure that accidental refreezing did not occur

• DNA quality or amount may have been insufficient. Determine appropriate lipid-to-DNA ratio empirically

• Cell medium may be contaminated with serum or antibiotics. Be sure that medium contains no growth enhancers or other additives

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

• Culture cells to ~90% confluency and use fresh, clean growth medium

2.  Prepare Transfectamine™ 5000-DNA mixture

• First mix 2.5 ug of DNA with 200 uL of serum-free medium.
• Add 7.5 uL of thawed Transfectamine™ 5000 to DNA/medium mixture.
• Mix well and incubate at room temperature for 20 minutes.

3.  Add Transfectamine™ 5000-DNA mixture to cell culture

4.  Culture overnight

5.  Analyze transfection efficiency with appropriate method

• Maximum expression levels can be discerned 72-96 hours post-transfection
• If using Flow Cytometry, Laser-Scanning Molecular Imaging, Microscopy, or Western Blot Analysis, a fluorescent label is necessary or strongly recommended

There are multiple ways to improve your transfection results with a particular cell line and reagent.

We suggest the following:

• Determine cell line transfection resistance

• Older reagent formulations have longer protocols, but more documentation of cell line compatibility

• Suspended cells often require higher densities than adherent cultures

• Scientific support and assistance from reagent manufacturer

The general steps of ion-exchange chromatography procedure are as follows:

1. A sample with proteins to be purified is loaded onto the ion exchange chromatography column at a particular pH, which ensures that the proteins are ionized and soluble.
2. Charged proteins will bind to the oppositely charged functional groups in the resin, and uncharged impurities are wash away.
3. A salt gradient is used to elute and separate proteins. Proteins with stronger binding are eluted at a higher salt concentration. A pH gradient may also be used, but cautions should be taken to make sure proteins are stable and soluble.
4. Unwanted proteins and impurities are removed by washing the column.

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 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.

There are four main phases in the growth curve of normal cultured cells, which typically displays a sigmoid pattern of proliferation.

• Lag phase: At this stage cells do not divide. It is the period when cells are adjusting to the culture condition and preparing for the cell division.
• Log phase: It is also called logarithmic phase or exponential phase, when cells actively proliferate and the cell density increases exponentially. It is recommended to assess cellular function at this stage since the cell population is most viable. Cells are also generally passaged at late log phase, because passaging cells too late can lead to overcrowding, apoptosis and senescence.
• Stationary phase (or plateau phase): Cell proliferation slows down due to a growth-limiting factor such as the depletion of an essential nutrient and/or the formation of an inhibitory product, resulting in a situation in which growth rate and death rate are equal. Cells are most susceptible to injury at this stage.
• Death phase (or decline phase): Cell death predominates at this phase and the number of viable cells reduces.

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.

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.

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:

• immunofluorescence is commonly used to stain microbiological cells
• immunohistochemistry is commonly used to stain sections of biological tissue
• immunocytochemistry is commonly used to stain intact cells removed from extracellular matrix

Bacteria and viruses differ in many ways, such as structure, size, pathogenicity as well as response to medications.

• Structure: Bacteria are single-celled, living organisms, which have all the components necessary to survive and reproduce outside a living host. Viruses have no cell wall or organelles, who are described as “organisms at the edge of life”. Although viruses have genes, they do not have a cellular structure to perform their own metabolism, thus requiring a host cell to make new products.
• Size: Bacteria are about 1000 nm in size, which are visible under light microscope. Viruses are much smaller, usually about 20-400 nm, which can be visualized by electron microscope.
• Pathogenicity: Most bacteria are beneficial for our good health and the health of earth’s ecosystems, with only 1% of bacteria causing diseases. Most viruses cause diseases.
• Response to medications: Antibiotics may be used to treat some bacterial infections, but they do not work against viruses. Antivirals, on the other hand, are engineered to treat viral infections, which are not effective against bacteria.

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.

• Irreversible inhibitors react with enzyme and can chemically change it by forming new covalent bonds. The key amino acid residues needed for enzymatic activity are often modified by these inhibitors, thereby the inhibition cannot by reversed.
• Reversible inhibitors, in contrast, attach to enzymes with non-covalent interaction, such as hydrogen bonds, hydrophobic interaction and ionic bonds. These inhibitors generally do not undergo chemical reactions and can be easily removed by dilution or dialysis.

Restriction enzymes are generally categorized into four groups, types I, II,III and IV, which differ primarily in structure, cofactor, cleavage site and specificity.

• Type I enzymes: These enzymes cleave at sites remote from a recognition site, which require both ATP and S-adenosyl-L-methionine as cofactors to function. They are multifunctional proteins with both restriction digestion and methylase activities.
• Type II enzymes: They cleave within or at short specific distances from a recognition site. Most of the enzymes fall into this group require magnesium. They are single function protein with only restriction digestion activity.
• Type III enzymes: These enzymes cleave at sites a short distance from a recognition site. ATP is required for this type of enzymes to function. They exist as part of a complex with a modification methylase.
• Type IV enzymes: They recognize and cut modified DNA, typically methylated, hydroxymethylated and glucosyl-hydroxymethylated DNAs.

There are at least four distinct types of introns:

• Group I introns: They are large self-splicing ribozymes that can catalyze their own excision from mRNA, tRNA and rRNA precursors. The secondary structure of group I introns has a nine-looped stem, which is required for splicing.
• Group II Introns: They are also self-splicing ribozymes that are found in rRNA, tRNA and mRNA, but they cut themselves differently than group I introns. Besides, the splicing of group II Introns involves the formation of a lariat structure.
• Nuclear pre-mRNA Introns: They are found in the nucleus in protein-coding genes that are removed by spliceosomes.
• Transfer RNA introns: They are found in tRNA genes and need proteins (enzymes) to be removed.

There are 4 types of reversible enzyme inhibitors.

• Competitive: A competitive inhibitor and the substrate cannot bind to the enzyme at the same time. This kind of inhibitors often has very similar structure with the real substrate of the enzyme. The inhibition can be overcome with substrate concentration.
• Uncompetitive: An uncompetitive inhibitor binds only to the substrate-enzyme complex, which inactivates the enzyme-substrate complex. This type of inhibitor is most effective when the substrate concentration is high.
• Non-competitive: A non-competitive inhibitor binds to a site other than where the substrate binds; therefore, it can bind to both the enzyme and enzyme-substrate complex. The binding of substrate is not affected, but the catalytic efficiency of the enzyme is reduced by the inhibitor. Since the binding of substrate remains unchanged, this inhibition cannot be overcome with high substrate concentration.
• Mixed: A mixed inhibitor also can bind to both the enzyme and the enzyme-substrate complex. However, the binding of inhibitor can affect the binding of substrate by changing the conformation of the enzyme. This type of inhibition can be reduced but not eliminated by increasing concentration of substrates.

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.

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 staining solution is made of X-gal (200 mg/ml), MgCl2 (1M), K ferri-cyanide (50mM), K ferro-cyanide (50mM), and PBS.
• The cells are then fixated in formaldehyde/methanal (37 %), glutaraldehyde (25 %), and PBS.

The steps for lacZ staining are:

1. Wash cells in PBS
2. Add fixative solution
3. Incubate for 2 minutes
4. Wash 3 times with PBS
5. Add staining solution
6. Incubate overnight at 37°C

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.