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Immunoprecipitation Kits on ZAGENO

Immunoprecipitation, (such as ChIP), is a method of extracting a specific protein out of solution with a complementary antibody. That is, each antibody works as a specific ‘hook’ that captures its designated protein. When antibodies are attached to a substrate and introduced to the protein sample, those 'hooks' will catch and hold onto their specific protein, allowing the protein to be purified (via the substrate) from its original source; a key step in Sample Preparation.

Scientists use chromatin immunoprecipitation (ChIP) to determine the location of the DNA binding sites for a particular protein. In understanding DNA–protein interactions, scientists can answer questions about cell replication, gene expression, and the role molecular structure plays in cellular function.

Expand your understanding in our Knowledge Section. Perfect your experiments with our Troubleshooting Guides. Join in the discussion within our Community. Learn about ZAGENO at How It Works.

Immunoprecipitation

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.

Chromatin

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.

Chromatin Immunoprecipitation

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.

Compare ChIP Kits

With our comparison engine, you can avoid all the time and energy wasted sifting through multiple web pages from different suppliers. At ZAGENO you can clearly see kits side-by-side, with the relevant attributes for each kit neatly in line for easy selection of the best product for you.

Click on the comparison below for a clearer view!

For example, this comparison shows:

  • the ZYMO-SPIN™ CHIP KIT from Zymo Research can target modified DNA.
  • the EPITECT CHIP ONEDAY KIT from Qiagen can be used before carrying out real-time PCR.
  • the IMPRINT® CHROMATIN IMMUNOPRECIPITATION KIT from Sigma-Aldrich is the cheapest per reaction.

The compare feature clearly exemplifies how every kit has strengths and weaknesses. Depending on what features you require for your experiment, these details should enable you to make an informed decision on the perfect kit.

The ZAGENO comparison does not highlight one kit to be better than the other, as the kit of choice may vary between researchers - depending on each individual's preferred attributes. The best kit is the one that meets your needs - ZAGENO allows you to make an informed decision with minimum effort.

Check out our How It Works page for a guide to using the comparison function.

Video and Image Credits

Video: SCI HELP/YouTube

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