Co IP with myc and ha tagged fusion proteins - (Jul/19/2005 )

Hi, I am trying to do a co-IP with HA and Myc--tagged proteins. I tried to pull down the Myc-tagged protein first, and could pull down ca. 5% of my Input. When I did the co-IP experiment, I can not detect my Myc-tagged protein anymore, there are no signs for degradation, and it is not in the supernatant.

The experiment was set up in this way:
Extracts were made from HEK 293 cell.
1. HA and Myc vector alone
2. HA-Protein 1 and Myc vector alone
3. HA and Myc-Protein 2
4. HA-Protein 1 and Myc Protein 2
Buffer
50mM Hepes
150mM NaCL
0,1% NP-40
1mM EDTA
1mM NaF
1mM Na3OV
1mM PMSF
10% Glycerol
pH 7,5
I added a Protease Inhibitor cocktail.

I lysed the cells for 20 min on Ice, then centrifuged for 20 min at 4°C.

For the IP I added approx. 2 µg of Myc monoclonal AB(Clontech), incubated 4 hours on a rotating wheel in the cold room. Then I added 20µl Protein G-Plus agarose (Santa Cruz). After 2 hours with the beads I spinned them down (1 min 1000rpm), aspired the supernatant, washed 3 times with 500 µl of Lysis buffer, the resuspenden the beads in 20 µl 2x loading buffer, boiled and run the Gel.
The proteins show strong colocalization, and in a yeast-2-hybrid they interact strongly.
Any suggestions?
Thank you

Flo

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Hi floeich,

you mentioned "no protein degradation" happened to myc fusion protien, but you also said " it is not in the supernatant".
Have you monitored the myc protein throughout the whole IP process? I mean:
1: cell lysis supernatant
2: supernatant you discarded after adding G-Plus agarose beads and incubation.
3: first round of wash solution that you discarded
4: Beads after final wash

For the abover 4 samples, you may need to do simple western to find out where your myc protein is.

Good Luck:)

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Yes, I monitored. It really disappears. I blotted again with a second antibody against the construct, it is simply gone. Transfer worked, as I can see the marker transferred quite good, and ponceau-staining shows proteins.
I think I go searching a hole to dig in and cry......

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you mean it disappears from the very first step?
In my previous post I listed 4 samples, if you even didn't find protein in sample 1, that suggest your myc fusion protein was in pellet.
good luck:)

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Hi again, of course it appears in sample 1, the cell lysis supernatant. But in the other three its away...
Sample 3 I did not do...I will test it. But the protein concentration will be very low, so I have precipitate with TCA or should I just load as much of the wash as I can?

Thx

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sample 3 is not as important as the others, unless you used very stringent washing buffer. But since you are doing an IP study, you won't introduce such washing buffer, will you?

Well, if the majority of myc protein was not in post-bind, elute and beads sample, then I would like to say your protein had been degraded. Besides PMSF and protease inhibitor cocktail, you need to look for additional protease inhibitors.

You can also check those journals in which your protein of interest was studied, figure out the protease inhibitor been used.

Good Luck

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Hi again,
now it worked, it seems that the AB was not working properly.I switched to another lot, and now I IP about 5-10% of my input. Should it be more?

Unfortunately my protein is a novel one which was not studied before, so I have to find all its properties regarding which kind and which amount of detergent are suitable.
Thx
Florian

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You are right when you say you should find out the conditions of lysis.
because actually I think 0.1 % NP-40 is very litle. I didn't even know that such few amount of detergent without sonication would be enough to lysate cells proprerly.

Usually people start with 1 or 0.5% NP-40. If they don't get anything IP protein. Then they start decreasing the % of detergent.

Starting with very low amount you risk not to lyse cells well enough and therefore not geting IP protein (it can stayed arrested in the nucleus, if is expressed in there) or you can have background/unspecific binding.

Did you do the negative control. Using agarose beads in the cell lysate but not using Ab to IP?

Glad you found out the Ab was the problem.

Good luck

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