Immunoprecipitation protocol

Go to the profile of Chinmaya Sadangi
Feb 06, 2018
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Hi,

I am doing a coIP, but I am stuck on a step with the protocol. The protocol is as follows:

Washing & Usage of Gamma binding beads

In a falcon tube, take 15 ml 1x (FS) PBS.
Take the Gamma-bind Sepharose beads from the fridge, shake it gently.
Cut a 1 ml pipette tip at the edge and pipette 100 µl of beads (for 2 tubes) from the stock and place it at the bottom of the PBS falcon tube. You need to adjust the number of beads to be taken, according to your experiment.
Spin the tube containing the beads in PBS at 2000g for 2 min.
Use a serological pipette to carefully remove the PBS until you reach 1 ml. Once you reach the 1 ml mark, use a fine tip (tips used for loading SDS-PAGE gels), insert the tip into the beads and remove the PBS until it's dry.
Add 1 ml of IP buffer to the beads, spin down the tubes at 2,000 g for 2 min and rotate it for 5 min. (Incubate on ice, when not on shaker).
In the last step (#6), should I discard the supernatant as the next step is to add the beads to antibody-lysate complex?

Should I try to scoop the beads from the bottom of the tube or should I take ~ 40-50 ul of the beads with the IP buffer and add it to my ab-lysate? If I use 40-50 ul, the rest of the beads with IP buffer can be discarded or can I store it at 4 degrees and use it for other experiments?

Thanks.

3 Comments

Go to the profile of relaxin
relaxin 5 months ago

Your protocol is to wash the beads with IP buffer. So discard the supernatant after washing.

It will be easier to resuspend the beads with IP buffer and then pipet the suspension to Ab-lysate. If you have leftover, you have to discard it unless you have other use next day. Fungus may grow at 4 C in the absence of preservative. To eliminate waste, calculate the amount you need for the experiment.

Go to the profile of Chinmaya Sadangi
Chinmaya Sadangi 5 months ago

Thank you Relaxin. I have been asked to suspend the beads in 1 ml of IP buffer. I use 40 µl of beads per sample.
If I mix the beads in IP buffer I don't see the beads as it takes time to settle. So, I just add 40 µl of the beads in IP buffer to the samples. If I leave the IP buffer for 30 min, I find that the beads are settling down and nothing was transferred.

So, what is the ideal way to transfer the beads?

Go to the profile of relaxin
relaxin 5 months ago

That depends on the amount of beads in the 1 ml suspension. If the amount of beads is low, you will not see any beads in 40 ul.

You should calculate the amount of beads you need for all samples (for 10 samples, you will need 440 ul). Mix the original stock well and pipet the amount you need and transfer to another tube. Then wash the beads and resuspend them in the original volume of IP buffer. Then pipet 40 ul of the suspension to each sample.