7 replies to this topic

[cellgene]

Hello

I am trying to show a co-immunoprecipitation.
I had a problem that I also had some of my protein in "only beads" control (without antibody) and also  I repeated with preclearing for 30min at 4degrees and although very weak, still I could observes someting in this negative control as well.
Now I am thinking of doing a preclearing overnight, has anybody tried this and is it recommended? would it cause any problems?

Also, do you ever make the IP longer than overnight? (2 days maybe?) As I don't really want to come to lab both on saturday and sunday  

thanks

[sgt4boston]

Why don't you do the pre-clearing over the weekend and try various types of blocked particles stringently washing the particles after blocking step?

This should not introduce any foreign proteins and remove most non-specific binding.

[cellgene]

sgt4boston, on Apr 23 2010, 06:30 PM, said:

Why don't you do the pre-clearing over the weekend and try various types of blocked particles stringently washing the particles after blocking step?

This should not introduce any foreign proteins and remove most non-specific binding.

thanks for the suggestion. So you think it is ok to incubate the lysate and beads over the weekend?
and I didn't understand what you mean by "various types of blocked particles" ?

[rkay447]

Your preclearing should definitely be extended to at least four hours, if not overnight.  You might want to think about doing more than one round of preclearing as well.  When I have major background issues, I've precleared up to three times.  I'll put in the beads, let it go for four hours, spin and put the lysate with fresh beads, let it go overnight, the next morning spin and put the lysate with more fresh beads for a few more hours.  You probably don't have to go to this extreme since you saw a reduction in the negative control with just one preclearing for 30 mins.  You could also increase the amount of beads you are preclearing with.  This will bind more of the nonspecific protein and let you preclear better, faster.  You can IP for longer times (2 days) but it isn't going to increase the binding and may result in your proteins starting to degrade.  I would recommend you add an excess of protease inhibitors and phosphatase inhibitors but you may still find your proteins degrade.   It might be fine though, the only way to know is to try.  Just realize that this longer incubation does increase non-specific background so if you are already having problems it may not be your best option.  Are you blocking your beads before incubating with lysate?  This will also greatly help background.  I always block in filtered 5%BSA/PBS overnight at 4 degrees.

[cellgene]

rkay447, on Apr 23 2010, 08:35 PM, said:

Your preclearing should definitely be extended to at least four hours, if not overnight.  You might want to think about doing more than one round of preclearing as well.  When I have major background issues, I've precleared up to three times.  I'll put in the beads, let it go for four hours, spin and put the lysate with fresh beads, let it go overnight, the next morning spin and put the lysate with more fresh beads for a few more hours.  You probably don't have to go to this extreme since you saw a reduction in the negative control with just one preclearing for 30 mins.  You could also increase the amount of beads you are preclearing with.  This will bind more of the nonspecific protein and let you preclear better, faster.  You can IP for longer times (2 days) but it isn't going to increase the binding and may result in your proteins starting to degrade.  I would recommend you add an excess of protease inhibitors and phosphatase inhibitors but you may still find your proteins degrade.   It might be fine though, the only way to know is to try.  Just realize that this longer incubation does increase non-specific background so if you are already having problems it may not be your best option.  Are you blocking your beads before incubating with lysate?  This will also greatly help background.  I always block in filtered 5%BSA/PBS overnight at 4 degrees.

Thanks for the suggestions!
I have never blocked the beads, or heard it from someone else in the lab. I might try it next time if this doesn't work.
I have been preclearing for 2 days Now I will  leave for IP overnight. I hope it will be fine.
I hope the proteins are not degraded... the excess protease inhibitors is also a good idea but we use "Complete Protease Inhibitor Cocktail Tablets", so we just add 1 tablet in 50ml of our lysis buffer. So I can't just add a little bit more of protease inhibitors... But maybe I could try adding a tablet to 40ml lysis buffer or something... Anyway, I will see tomorrow

[cellgene]

After 2 days of preclearing and overnight IP, background is very much decreased, with very short exposure(2 seconds- with camera) it is not there, however if I do it a little bit longer the band appears. But this would be enough, right?

Thanks everyone for suggestions,
if I repeat it maybe I will try changing the beads during preclearing...

[rkay447]

cellgene, on Apr 27 2010, 09:17 AM, said:

After 2 days of preclearing and overnight IP, background is very much decreased, with very short exposure(2 seconds- with camera) it is not there, however if I do it a little bit longer the band appears. But this would be enough, right?

Thanks everyone for suggestions,
if I repeat it maybe I will try changing the beads during preclearing...

Hey, as long as you are getting a band that only appears in the specific IP and not the control IP, you have a specific interaction.  I've had to do overnight exposures (film) but it's still valid data.  Glad to hear things are working out!!

[Mardahl]

@rkay447 (or others:-))
How do you expose a film overnight? Doesn't something (I don't know where - perhaps the blot itself) go bad? If I am seeing a clear signal in my input but a weak signal in my IP lane, could this mean that I should expose overnight? I would think not, since my protein is very clear in the input (it'd get overexposed I presume).

I have to add to this topic by saying that I have problems with protein precipitation during my clearing of the lysate before putting it onto the beads and I ALWAYS use fresh lysate, never freeze it at any point.

Any thoughts on this?