How many cells is an IP?
Generally, I use between 106 and 2×107 cell equivalents for each precipitation, depending upon the expected expression level.
How do you know how much antibodies your IP address is?
For best results, the optimal amounts of antibody should be empirically determined. But a general rule is to add 2 to 10 micrograms of antibody per 500 micrograms of lysate. If you are using neat antisera, or an IgG fraction (such as protein-A purified antibody), greater amounts of antibody are likely to be required.
What is a co-IP?
Co-immunoprecipitation (co-IP) is a popular technique to identify physiologically relevant protein–protein interactions by using target protein-specific antibodies to indirectly capture proteins that are bound to a specific target protein.
How many cells do you need for immunoprecipitation?
Too many cells/too much protein in lysate leading to a lot of non-specific proteins in eluate. Reduce the number of cells/lysate used . Generally using 10-500 µg cell lysate is enough .
What is lysate used for?
A fluid containing the contents of lysed cells is called a “lysate”. Cell lysis is used to break open cells to avoid shear forces that would denature or degrade sensitive proteins and DNA.
How much protein do you need for IP?
Use 25 µl of Protein A or Protein G Magnetic Beads per 200 µl of crude cell lysate containing 200-500 µg of total protein in a standard immunoprecipitation protocol.
How much protein should I add to my IP?
Protein extract should not be too dilute to avoid loss of protein and to minimize the sample volume to be loaded onto gels. The minimum concentration is 0.1 mg/mL; optimal concentration is 1–5 mg/mL.
How much protein do you need for co IP?
You want to start with a fair amount of material; aim for between 1 and 3 mg of total protein for every 0.2-0.5 ml of your starting sample volume. You should also aim to keep your target protein as happy as possible throughout the disruptive procedure of cell or tissue lysis.
What is the difference between immunoprecipitation and Coimmunoprecipitation?
In immunoprecipitation (IP), an antibody is used to purify its specific target, or antigen from a mixture. In co-immunoprecipitation (Co-IP), an antibody is used to purify its target antigen, along with its binding partners, from a mixed sample.
How do you lyse cells for co-IP?
For Co-IP of soluble proteins, use a non-detergent, low-salt lysis buffer. This mild lysis buffer is probably least likely to interfere with protein-protein interactions. For less soluble protein complexes, add non-ionic detergents such as Nonidet™ P40 Substitute or Triton™ X-100 to lysis buffer.
How much antibody do I need for ChIP?
We recommend using 2-10 µg of your ChIP antibody depending on the abundance of your protein target and the affinity of your antibody for the target. More antibody does not always equal stronger signal. It is suggested that to get the best ChIP signal the amount of antibody be titrated.