Immunoprecipitation on Zageno
There are several types of immunoprecipitation, such as individual protein, RNA, or chromatin immunoprecipitation (ChIP); and different methods (direct or indirect) of performing these techniques, The direct method employs antibodies that are already attached to solid substrates, such as magnetic beads or agarose gels. The indirect method adds antibodies to a solution containing proteins, allows the solution to rest, then solid substrates like magnetic beads are added to bind to the antibodies. The indirect method is particularly useful for when protein concentration is low, or when the antibody affinity for proteins is weak. The direct method is the most commonly used in the lab.
One of the key uses for immunoprecipitation is studying the role chromatin plays in the cell. Chromatin is a molecular complex consisting of DNA, RNA, and protein; the structure of it, however, depends on the cellular phase. Nonetheless, chromatin serves to compact the genome into smaller segments to fit inside the cell, reinforce the DNA strand to allow mitosis, prevent damage to DNA, control gene expression, and aids in DNA replication.
Chromatin is formed via histones; small proteins which DNA wraps itself around to pack itself together. Chromatin can be lightly or tightly packed depending on whether the cell is in metaphase or interphase.
During interphase, the chromatin complex is loose; allowing DNA polymerase access to the DNA strand for DNA replication. Furthermore, genes which are actively transcribed are looser; genes that are not (i.e. turned off) are more tightly wound, preventing DNA polymerase access.
As mentioned previously, to study chromatin scientists employ chromatin immunoprecipitation, commonly known as ChIP. ChIP is a means to extract chromatin from the cell, purify the DNA, and sequence it to determine where proteins, such as transcription factors, bind to DNA. In knowing the genetic sequence of where these proteins bind to DNA, scientists can better understand gene regulation and expression, as well as cellular replication.
ChIP is based on the principle that DNA binding proteins can be cross-linked to their binding site. So, following the principles of immunoprecipitation, proteins are crosslinked to their complementary DNA strand (via formaldehyde) in vivo. Cells are then lysed, and DNA is broken to smaller strands via sonification (with the proteins still attached). Then, immunoprecipitation is employed, purifying the chromatin complex.
To denature the DNA-protein crosslink, heat is applied (around 70 °C). Then, what is left are small DNA chunks which can then be amplified in PCR for further analysis. One tricky aspect, however, is designing the correct primers for the unknown DNA region you just extracted. One way around this is to clone the DNA strand into a plasmid vector and use the known primers for that vector to replicate your purified DNA.
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Video: SCI HELP/YouTube