I have been performing CoIP experiments with the ThermoFisher Pierce classic Magnetic IP kit. I havent couple my antibody to the beads, instead incubated lysate, antibody and beads in a tube overnight at 4oC on a rotator.
The next day I have collected supernatant samples and tried various different wash steps/combinations with buffers of 150 mM NaCl and buffers of 500 mM NaCl. However with all combinations of washes, I am getting unspecific bands at 150 and 200 kDa as well as the bands at 50 and 25 kDa for the heavy and light chains of anitbodies.
Can anyone suggest what these bands could be? Or what optimisation steps may be successful at minimising them?
Dear Alice
You can add a preincubation step, you mix clean beads and the lysate for 4 hours in a rotator. Then you collect the supernatant and follows the same CoIP protocol that you use. You can also lower the amount of protein used in your current CoIP protocol.Hope it helps.
hi
1- wash the protein A/G beads before use (2 times with 500ul wash buffer )
2- centrifuge cell lysate after deffrizing (13000 rpm 10min 4 c)
3- incubte cell lysate with protein A/G beads for 1 hour at 4c( capture free IgG in cell lysate )
4- remove supernatant from beads
3- incubate supernatant + primary antibody ( overnight 4c)
4- incubate cell lysate + antibody with protein A/G beads ( 1 hour 25 c)
# finally run the sample under non reducing condition in WB (omit DTT or 2ME from sample buffer )
good luck
a. could be degradation or clumping of you protein.
Keep them cold, and work quickly. Decrease lysate incubation time.
b. add another wash step after binding to beads to column.
c. lower the amount of protein used on western blot.
d. lower amount of protein in co-ip
e. cut out this section of PVDF membrane before incubation wit primary antibody.
run a ladder on first and last well, line up with ruler and cut via razor. Mark the sides so you can puzzle peace it back together for image.
Hi, just wondering if there is any reason for incubating over night? often, extending the incubation time will result in increased background. in many/most cases a shorter incubation time (as short as 10-15 min) can be sufficient both to load the Ab to the beads and to pull out target from the lysate. Elimination of the light and heavy chain bands from the blot can be done by using the TrueBlot HRP conjugated secondary Abs. kind regards ketil
Hello,
Just to add to the previous answer,
1- If you don't use a reducing agent in your loading buffer, you might get a band above 100 kDa .
2- Try to avoid using antibodies from the same species. For instance, use mouse antibody to IP and rabbit antibody to blot. This way you will avoid the heavy background caused by IgG
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