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Immunoprecipitation on Zageno

ChIP-IT® Protein G Magnetic Beads Active Motif
Targets / Sample Amount / Technology Protein G conjugated magnetic beads
From $ 250.00 (25 Reactions)
Sizes 1 (25 Reactions)
Catalog IDs 53014
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ChIP-IT® Express HT Active Motif
Targets / Sample Amount 1-50 µg chromatin Technology Protein G conjugated magnetic beads
From $ 1,050.00 (96 Reactions)
Sizes 1 (96 Reactions)
Catalog IDs 53018
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Classic Immunoprecipitation G-Biosciences
Targets Proteins Sample Amount / Technology Spin column
From $ 256.00 (50 Reactions)
Sizes 1 (50 Reactions)
Catalog IDs 786-637
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EpiNext™ ChIP-Seq High-Sensitivity Kit Epigentek
Targets / Sample Amount 1 x 10^5 to 1 x 10^6 cells, 5-50 mg tissue Technology ChIP-Seq
From $ 539.00 (12 Reactions)
Sizes 2 (12 - 24 Reactions)
Catalog IDs P-2030-12, P-2030-24
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EpiTect ChIP OneDay Kit QIAGEN
Targets Genomic DNA Sample Amount 1 x 10^6 cells Technology ChIP
From $ 294.00 (12 Reactions)
Sizes 1 (12 Reactions)
Catalog IDs 334471
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Pierce™ Direct IP Kit Thermo Scientific
Targets / Sample Amount / Technology IP
From $ 312.00 (50 Reactions)
Sizes 1 (50 Reactions)
Catalog IDs 26148
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EZ-ChIP™ Merck Millipore
Targets Genomic DNA Sample Amount / Technology Chromatin Immunoprecipitation
From $ 477.00 (22 Reactions)
Sizes 1 (22 Reactions)
Catalog IDs 17-371
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Magna ChIP™ G - Chromatin Immunoprecipitation Kit Merck Millipore
Targets Genomic DNA Sample Amount / Technology Chromatin Immunoprecipitation
From $ 413.00 (22 Reactions)
Sizes 1 (22 Reactions)
Catalog IDs 17-611
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Imprint® Chromatin Immunoprecipitation Kit Sigma-Aldrich
Targets Genomic DNA Sample Amount / Technology Chromatin Immunoprecipitation
From $ 282.50 (24 Reactions)
Sizes 2 (24 - 96 Reactions)
Catalog IDs CHP1-24RXN, CHP1-96RXN
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Pierce™ Classic IP Kit Thermo Scientific
Targets / Sample Amount 10 mg Technology Immunoprecipitation (IP), Affinity Purification
From $ 307.00 (50 Reactions)
Sizes 1 (50 Reactions)
Catalog IDs 26146
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Pierce™ Co-Immunoprecipitation Kit Thermo Scientific
Targets / Sample Amount 10 mg Technology Co-IP, Affinity Purification
From $ 362.00 (50 Reactions)
Sizes 1 (50 Reactions)
Catalog IDs 26149
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Direct Immunoprecipitation Kit G-Biosciences
Targets Proteins Sample Amount / Technology Immunoprecipitation
From $ 256.00 (50 Reactions)
Sizes 1 (50 Reactions)
Catalog IDs 786-636
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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.

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.

Video and Image Credits

Video: SCI HELP/YouTube