The problem you must know in Chip experiment!

Veröffentlicht von: ftpglobal
Veröffentlicht am: 22.01.2018 07:37
Rubrik: Handel & Wirtschaft


(Presseportal openBroadcast) - 1. Do you know the difference between Immune precipitation process agarose beads and magnetic beads, which kind of Beads better?

Agarose beads have the advantage of being inexpensive in the early literature, because of inexpensive, but they were centrifuged for a long time, and the beads could break during centrifugation. Sepharose beads have non-specific binding and therefore require "pre-washing" of chromatin to remove proteins or DNA that may not specifically bind to protein G-agarose. Also, the stratification is not obvious; the sample is easy to lose.

The current mainstream method is the beaded method, the advantage of beads is the sample without centrifugation; the operation time is short, the other bead surface is smooth, the background is low, so no need to close. Another advantage of magnetic beads is colour, layered clear, the sample will not be lost, but the magnetic beads need to be a magnetic frame. Therefore, immunoprecipitation is the recommended method of selection of magnetic beads.

Agarose beads and magnetic beads schematic

2: When immunoprecipitation, sample, antibody and bead loading and reaction sequence on the experimental results have any effect? Is there any requirement?

There are generally three kinds of reaction sequence:

1.Chromatin sample + antibody first react, and then add beads;

2.Antibody + magnetic beads first react, then add the chromatin antibody;

3.Chromatin sample + antibody + magnetic beads three simultaneous reactions.

Whichever bead you choose, the order in using beads or agarose beads may affect your ChIP signal.

The first method involves incubating the beads with the capture antibody (hours at room temperature or overnight at 4 ° C), followed by the addition of chromatin (sample) and continued incubation (1 hour to overnight at 4 ° C with shaking). Increasing the incubation time is likely to increase background and ChIP signal; however, antibodies with low affinity to the target did not produce significant ChIP signals even after prolonged (overnight) incubation.

In the second method, the antibody and the chromatin sample are incubated together first, and then adding the microbeads can also obtain the result similar to the first reaction sequence, which can perform the immunoprecipitation well. However, it is not advisable to add these three components simultaneously for immunoprecipitation. Experiments have shown that the simultaneous addition of the three elements reduces the time required to carry out the entire reaction, but results are inferior to those of the above two methods.

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