An Overview of Biochemistry Lab Techniques

Proteins

First, let’s dive into the common tests used to analyze proteins.

Western Blot

The Western Blot is a ubiquitous technique used to qualitatively detect the prevalence of protein in a sample. To detect this, a select sample is digested, run on a gel, and treated with an antibody against a target protein. As depicted in the blot above, a household gene, such as β-actin or gap GH, is used as a control since they are typically expressed uniformly across the sample. Abnormalities in these bands can indicate issues with the blot and it may be beneficial to repeat the Western Blot for credible data. Interpreting Western Blots, are often more subjective than one would imagine: the intensity of the bands indicate the level of protein expression—the stronger the band the more protein; however, it’s important to note that these bands can only be compared to the samples it was run with, and not to external blots.

Antibodies

Antibodies are y-shaped proteins that recognize antigens (both pathogenic and nonpathogenic) and have a widespread role. For example, antibodies are used in Western Blots to bind to the target proteins. Other variations of antibodies, such as scFvs, nanobodies, and camel antibodies have similar roles and can be used for labeling, especially in in vivo studies.

Co-Immunoprecipitation and Pull Down Columns

To study protein-protein interactions, researchers may use co-immunoprecipitation or pull down columns. The core concept is that when a researcher pulls out the target protein and the complex proteins that are associated with it, they can identify either known or novel interactions. In Co-immunoprecipitation, an antibody against a known protein is inserted and a certain kind of bead is used to extract the antibody and the bound proteins. A western Blot can then be run to further analyze the proteins. A pull down column, such as the GST pulldown assay, works similarly. When lysate is added to the column, GST (glutathione s-transferase) tagged bait protein attaches to Glutathione resin (immobilize glutathione) while unbound proteins are eluted. This results in only GST tagged protein and associated/interacting proteins bound.

Biotin and Streptavidin

Some of the most common bait tag systems used to accomplish this are Biotin and Streptavidin since they form one of the strongest bonds in biology (KD ~ 10-14). This strength can be exploited by creating “Biotinylated” proteins or antibodies that will bind to streptavidin beads when pulled out. To further control the kinetics of the reaction and prevent multiple Biotinylated sites, protein engineering and “avi-tags,” containing one specific, well defined biotinylated site, is often used.

IMMUNOFLUORESCENCE

Immunofluorescence is an extremely common and general term in biology which refers to a fluorescent tag (such as GFP) conjugated to an antibody, used to identify a protein of interest. It's useful for showing where proteins colocalize.

ELISA

ELISA are used to detect the presence of a certain antigen (like the COVID-19 rapid test) through the use of antibodies that process a substrate. Direct ELISAs is when antibodies bind to bound antigen, so the conjugated enzyme processes substrate. Indirect ELISAs involve primary antibody binding to antigen and secondary antibody binding to the primary. Sandwich obtains capture antibodies which binds antigen, followed by secondary antibody, and finally competitive ELISAs employ antibodies that binds to sample antigen while free antibody (that do to sample antigen) can bind to plate-bound reference antigen.

Similar to qPCR, standard curves are required for quantitative absolute antigen measurements. Optical Density (a measure for change in color) can detect absorbance, as ELISA enzymes produce color relative to antigen concentration.

Cytometry Time of flight is a more novel technique which can measure cell populations through antibodies bound to metal ions. As opposed to fluorescent flow antibodies, this results in a decreased issue with signal overlap or background signal and corresponds to mass charge ratio, allowing it to be used to measure abundance.

Nucleic Acids

Now let's shift gears and discuss nucleic acids.

Polymerase Chain Reaction

There are a variety of PCR that can be run, but all essentially share the use of complementary DNA fragments called primers and involve a target DNA region to be amplified over successive rounds of DNA polymerase.

Quantitative PCR (qPCR) assesses DNA quantity over rounds of PCR, enabling the measurement of DNA present at the beginning. This is measured by intercalating fluorescent dyes, such as SYBRgreen, or specific hybridizing probes, like TaqMan, and the more fluorescence that results correlated to the higher expression of the target DNA.

This data can be graphed on an Amplification plot which assesses the relative fluorescence of samples over successive rounds of pPCR; however this is only useful for relative gene expression. A Standard curve enables the absolute DNA quantity measurement by building a curve with known DNA concentrations. Finally, melt curves determine the specificity of qPCR.

Molecular Cloning can be accomplished by first making a plasmid. Restriction enzymes perform double stranded breaks at specific sequences, enabling the insertion of a fragment into another, such as an expression plasmid. Most will often leave overhanging, “sticky” ends that enable easier insertion of complementary fragments.

Various techniques can be employed to determine the sequence of nucleotides in a DNA sample. Sanger sequencing, utilizes fluorescent dideoxynucleotides to generate fragments which are then run on a gel, while a chromatogram is used to assess sequence quality.

A more novel approach is Next Generation Sequencing, or a catch-all term for sequencing methods that are fastened or simply better than Sanger sequencing. There are many other sequencing methods and techniques we can delve into.

With the advancement of techniques from Western Blots to Polymerase Chain Reactions, the efficiency and scope of research has significantly increased, fostering the development of new discoveries and studies in a streamlined manner.