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Immunoprecipitation protocol

coIP immunoprecipitation western blot

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#1 Mad Researcher

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Posted 29 January 2018 - 11:50 AM

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 

  1. In a falcon tube, take 15 ml 1x (FS) PBS.
  2. Take the Gamma-bind Sepharose beads from the fridge, shake it gently.
  3. 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. 
  4. Spin the tube containing the beads in PBS at 2000g for 2 min.
  5. 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.
  6. 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.


Cheers,

Mad Researcher

#2 rkay447

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Posted 05 February 2018 - 02:11 PM

I think the final step is to equilibrate the beads in your lysis buffer.  I would add the IP buffer, rotate and then spin.  Remove as much of the IP buffer as possible without disturbing the bead pellet.  Add a fresh ml of lysis buffer, resuspend beads evenly and then pipette the bead slurry into your ab-lysate with a bit of the IP buffer.  You can save the beads at 4C for future experiments, but I would recommend the addition of about 0.05% sodium azide to prevent bacterial growth.  Future use would require washing the NaN3 out however.  You might be able to resuspend the beads in a 50-50 mix of IP lysis buffer/ethylene glycol and store at -20C.



#3 Mad Researcher

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Posted 05 February 2018 - 04:24 PM

I think the final step is to equilibrate the beads in your lysis buffer.  I would add the IP buffer, rotate and then spin.  Remove as much of the IP buffer as possible without disturbing the bead pellet.  Add a fresh ml of lysis buffer, resuspend beads evenly and then pipette the bead slurry into your ab-lysate with a bit of the IP buffer.  You can save the beads at 4C for future experiments, but I would recommend the addition of about 0.05% sodium azide to prevent bacterial growth.  Future use would require washing the NaN3 out however.  You might be able to resuspend the beads in a 50-50 mix of IP lysis buffer/ethylene glycol and store at -20C.

Thanks rkay447.

 

I want to avoid adding the lysis buffer as it doesn't contain the detergent (the IP buffer does have detergent).

So, how much beads with lysis buffer should I add to the ab-lysate mixture? I generally add 40 µl. The confusion is/was should I take 40 µl of IP buffer-beads mix or should I scoop the bottom.

Sodium Azide is a great idea. I might use it in future. As of now, I store the beads in 25 µl of sample buffer mixed with DTT. I load this amount to the gel. How can I wash the sodium Azide?


Edited by Mad Researcher, 05 February 2018 - 07:24 PM.

Cheers,

Mad Researcher





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