Precipitate in IP

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Scarnati14
Scarnati14's picture
Precipitate in IP

 I keep having this reoccuring issue with my immunoprecipitations. I will give a brief rundown of how I do my IPs.(very simplistic overview)
- incubate with Ab for 1 hour~to overnight
- shake with protein A beads for 1 hour (25uL of 50% slurry)
- wash the beads
-add 2x loading dye with DTT(20uL)
- boil for 5 minutes
- quick spin at top speed
Now when I then go to load my gel I always seem to have a viscous precipitate at the bottom of my eppendorf tube. Does anyone have any ideas of how to get around this. 
Any feedback would be great.
THanks
-Scar

vanshita
vanshita's picture
What you mean by washing

What you mean by washing beads? instead of washing you can directly add loading buffer into beads and boil sample with the beads and after giving high centifuge load the supernatant.
one thing i want to ask why you are using DTT in loading buffer instead of SDS whc we normally use in buffer.

Chin Fen Teo
Chin Fen Teo's picture
 Hi Scarnati14,

 Hi Scarnati14,

Is it possible that the white precipitate that you observed comes from the beads? Or you think it is due to the IPed protein?

If it is the first case scenario, you may use a flat gel loading tip to transfer the IPed supernatant into a fresh tube so that you won't have to worry about pipetting the bead into the well.

If it is the second case scenario, you may heat the sample instead of boiling- I learnt from earlier post on this forum (the FFM), but never encounter such in my own hands, that certain proteins don't like being boiled and they tend to precipitate out during the boiling step. One can overcome by heating at ~70 deg.

Good luck.

P/S- To vanshita, adding reducing agent (DTT, 2ME etc) in the sample buffer (which already contains SDS itself) is a routine practice for IP experiment. As the name implies, it reduces the disulfide bonds, so that when the sample is resolved on the SDS-PAGE, the antibody shows fragments of 50 and 25 kD instead of 150 kD.

Scarnati14
Scarnati14's picture
 I think the precipitate

 I think the precipitate doesn't come from the beads themselves, it is precipitated protein. It is almost like boiling an egg, I feel, that maybe i just have so much protein that it tends to come out of solution. Someone suggested to me a while back to try increasing the volume of loading dye I add?
Have you ever heard of this?
-Scar

Chin Fen Teo
Chin Fen Teo's picture
 Hi Scarnati14,

 Hi Scarnati14,

What is the scale of IP are you working on? (Ab:lysate:bead:sample buffer) 

If you were using the standard amount that one can get from the company instruction manual, I don't see how can you have too much protein... But it is always worth a try. Before that, have you tried to lower the temperature as mentioned? 70 deg, 10 min should do...

Good luck.

vanshita
vanshita's picture
thanks pippuri.

thanks pippuri.

Scarnati14
Scarnati14's picture
 I will give it a shot thanks

 I will give it a shot thanks a lot

sunilragu17
sunilragu17's picture
WAsh steps are absolutely

WAsh steps are absolutely necessary since some proteins will be .left over during centrifuging the beads...
I suggest u boil sample for a lesser time as previously suggested by one of our colleagues here... Just load the complete vial of your IP sample along with beads.

Regards-
Sunil