Many methods in molecular biology are simply different combinations of a handful of techniques. These combinations can be represented as a “family tree” whose “trunk”/”backbone” is PCR (polymerase chain reaction) and whose “roots”/”foundation” is built upon: (1) chemical synthesis of short oligonucleotides, (2) fully sequenced genomes and (3) vectors derived from bacteria and viruses.
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Posted in Biology, Molecular Biology
While microscopic methods with protein and atom scale resolution – that don’t break the diffraction barrier! – exist (e.g. electron microscopy, atomic force microscopy, x-ray crystallography, etc.), they tend to be more practically difficult than fluorescence-based microscopy (see posts on Fluorescent Probes and Fluorescent Antibodies). Therefore, to give fluorescence microscopy nanometer resolution, several super resolution techniques have been developed that combine clever (1) optical/photophysical and (2) computational-processing tricks to “clean up” the “blurred data.”
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Posted in Biology, Cellular Biology
Basic light microscopy can only resolve objects that are larger than 100 nm which means that while it can visualize animal cells (~10,000nm), organelles and bacteria (~1,000nm), it cannot visualize viruses (<100nm), proteins (<10nm) or small molecules(~1nm) (see post summarizing Biological Scales). This limitation is known as the “diffraction limit” and is caused by the fact light only interacts differently with objects separated by more than one wavelength (λ). Intuitively, its helpful to think of each of these wavelengths as “a minimum pixel size” for a computer image where: infrared light (λ ~ 10.0μm) has pixels 100 times larger than ultraviolet light (λ ~ 0.1μm).
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Posted in Biology, Cellular Biology, Microscopy, Physics
In a follow-up to our post introducing ELISA, we wanted to discuss a common application of this technique: small molecule inhibitor screening. The set up is relatively simple (1) Coat a 96-well plate with your two proteins of interest and and a detector antibody (2) add a library of 96 molecules per plate (3) Inspect which molecules inhibit the protein-protein interaction (resulting in a color loss).
Once you have have narrowed down your library to a list of inhibitors, you can rank those inhibitors by their potency/IC50 by running dilution series with the exact same plate set-up:
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Posted in Chemical Biology, Chemistry
ELISA or Enzyme-Linked Immunosorbent assay is the most commonly used method for measuring proteins concentrations in solution. It is extensively used both in the laboratory (e.g. culture supernatant) and the clinic (e.g. blood tests) due to its simplicity and adaptability to other protein-based assays such as high throughput screening. The way it works is outlined in the figure above and described in detail below:
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Posted in Biology, Cellular Biology
Flow cytometry is a technique that can quantitatively measure (1) protein expression levels per single cell and (2) amounts of different cell-types (based on a protein-maker) for thousands of cells in minutes! Flow cytometers use hydrodynamic focusing to force a mixture of stained cells into a single-file line through a flow cell. Then, each cell, sequentially, passes through a laser beam which excites the fluorophore allowing quantification of protein expression for all proteins that are stained!
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Posted in Biology, Cellular Biology
Following up on our post introducing antibody-based experiments, we wanted to describe how antibodies are made in large volume: First, an animal is immunized with the protein (or peptide) of interest; Second, the spleen is dissected and the plasma cells producing the antibodies of interested are isolated; Finally, these plasma cells are fused with myeloma cells to “immortalize” them into a hybridoma (an antibody-producing cell-line that can be cultured indefinitely).
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Posted in Animal Work, Biology, Cellular Biology
Antibodies are immune proteins that can be easily engineered to bind (and “detect”) any given protein in via a simple vaccination method. To “signal” the presence of detected proteins, fluorophores are attached to “stain” the protein (and cell or tissue expressing it) a particular color. These fluorescent probes, have been utilized to identify the location of specific proteins in tissue sections (histology), single cells (microscopy) and quantify the amount of protein in cells (flow cytometry) and in complex mixtures (western blotting).
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Posted in Biology, Cellular Biology
Inbred mouse lines are used for most laboratory mouse work to make sure that all the mice have nearly identical genetic backgrounds (i.e. genetic code). These lines were originally generated by repeatedly mating mice with their siblings which minimized genetic variability and makes these mice as close to clones of each other as is practically possible.
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Posted in Animal Work, Biology
Careful breeding/husbandry of mice is important when one is asking genetic questions about disease. Such questions include: “Does gene X contribute to cancer?”, “Does my drug treat cancer by targeting gene X?”.
In general, these questions can only be answered by comparing mice that have gene X (WT or +/+) or don’t have gene X (KO or -/-). To make sure the genetic difference is the only difference between the mice, you usually compare siblings of inbred mouse-lines which have the same sex, age, environment and genetic background.
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Posted in Animal Work, Biology
Pronation describes the roll of the foot from “initial impact” (at the outer heal of the foot) to “toe-off” at the front of the foot.
Principal component analysis (PCA) attempts to find true trends hidden in complex data by filtering out noise and redundancy. It does this by treating complex data as a n-dimensional shape (where n is the number of measurements in your study) and fitting that shape to n 1-dimensional lines called: “principal components” and ranking these lines by the percentage of data variation that they capture.
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Posted in Biology, Systems Biology
For distance running there are three main factors that determine racing performance (or fitness): Oxygen transport (VO2max), Lactate Threshold (LT) and Running Economy (RE). The figures below, illustrate how training/de-training affects each of these factors over time:
Posted in Running
Have you ever wondered why your calves are sore after a 5k but your hamstrings are sore after a marathon? The reason is your calves bear more of your weight as you run shorter/faster and your quadriceps bear more of your weight as you run longer/slower.
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Posted in Running
Recently, while running a workout on a windy track, I started wondering about the science of air-resistance and drafting. More specifically, I wondered what difference was between 10 mph tail-wind and the 10mph head-wind I encountered on opposite sides of the track (see figure above). When I returned home, I discovered some classical papers that answered this exact question in an experimental and theoretical way (summarized in the Figure above).1-4
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Posted in Running
