7 replies to this topic

[Trof]

I'm new in proteomics and doing IP with A/G agarose beads, but I struggle with what is the best way to pipette them.

 

Here they don't resuspend the whole volume, just a bit and semi-randomly aspire 30 ul of partly mixed beads. This leads to uneven volume of beads in each sample and it's sometimes very difficult to pipette. And since I just remove all except the beads at the end of wash and mix with fixed volume of Laemmli, this leads to uneven final volumes also (we do not measure protein concentration, just use it as it is, so then uneven protein concentration).

 

I was trying to find ways to improve this, I got some information that mild vortexing the beads alone (before adding to lysate) would not damage them and evenly mix the whole volume so I can pipette it more evenly. Well, mostly, sometimes there are very littel beads.. but bigger problem is, vortexed beads will attach on the well walls and they won't go down even by spinning.

I decided to check visually the volume of beads before incubating, but still I wonder, what is the best trick to handle those.

Incidentally, the protocol used here incubates sample with Ab for 1-2 hours rotating, and then overnight (in coldroom) with beads before washes. I was one one day short last time and needed to shorten it up and manufacturer bead protocol said two hours of incubation could do it, so I tried 2 hours with Ab alone, 2 hours with beads. This IP ended up badly (this is a repeat of an successful one, so it should work ) and I wonder if shortening the incubation could have the effect (or beads damage since I did vortex them to mix, they are not very eager to). I try not to disturb the complexes by rough mixing of course.

 

This problem may repeat, since I'm kind of forced to finish even those experiments where I'm a day behind. So my option is either to IP in one day (not making bead incubation overnigh) or leave it in the rotator for the whole weekend (can't go to lab). Which do you think would be better option? (I still don't know if the shortening of beads incubation caused the experiment to fail, but it could).

I would be grateful for any other tips regarding work with the beads, thanks.

[mdfenko]

mild vortexing shouldn't affect your results. even if the beads break they will still bind the antibodies and the fragments will settle (broken beads will only have an effect on chromatography).

 

if you choose not to vortex then you can suspend the beads evenly by trituration (since you're not using all of the beads for the gel you don't have to worry about some sticking to the sides).

 

incubation time depends on the avidity of the antibody at the conditions you use.

 

incubation over the weekend should be okay as long as the system is protected from proteases (either with inhibitors or purified components). the only problem with this is reproducing the incubation in later experiments, if it's an issue.

[Trof]

Thanks, mdfenko,

I only use that lysate for WB, so in that case I guess vortexing it should not affect it.

 

Would you care to explain the trituration in detail, I think I never heard the word (and googling doesn't help that much)

 

Protease inhibitors are present in the buffer (cOmplete tablet) and AFAIK should be stable for that time (fresh made). 

 

The point about reproducibility is actually a good one, I don't have much experience with this, I know the stringency (that may apply to length of incubation and rotation and other things) can affect the binding partners. In my case I only care about two proteins cotransfected into cells, and they should be directly binding.
From what I was able to find, the process is intended to be rather stringent, we want just the direct binding partners. 


Now I was thinking the length of incubation will only affect the overall number of proteins binded, but also, the time can affect the complex stability. So shortened incubation would lead to decrease of overall protein binded (i.e. when I do WB for the IPed protein and his parner, there would be neither), and overextended incubation could lead to loss of the binding complex, so I would detect IPed protein only. Or have no effect at all. Am I right?

(edit: wait... I forgot, that after beads are added, the Ab binds to beads, yet at the same time it still continues to bind the specific complexes, the bead incubation also increases the overal Ab binding time...)

[bob1]

Trituration is pipetting up and down a few times. Often for IPs I use tips with the end cut off to help pick up consistent volumes without disrupting  the mix too much.

 

Length and temperature of incubation can play a role in antibody complexing - 4 C is more stringent than higher temperatures because of the activation energy required for the antibody to bind. Obviously longer incubations help increase the time for interaction between antibody and protein, but can also result in loss of interaction between two proteins. However, for demonstration on interaction, the proteins should already be in complex in the cell; you don't want additional interactions happening in the tube, and 4 C might prevent this to some extent.

[mdfenko]

in rereading your initial post, i noticed that you don't specify the temperature of the 2 hour incubation with the beads. if you incubated at 4C then that may be why you obtained a poor result. shorter incubations are usually performed at higher temperatures (25-37C) to encourage more rapid binding.

[Trof]

mdfenko: Yes, it was 4C. Thank you for suggestion. I had to repeat this shortened protocol this week again but this time with 4 hr incubation (4C).

I tried trituration mixing before adding to each sample with a new vial of beads and they seem more consistent this time, so it really is a better way. I just wondered... it ist possible to visually check the amount of beads before incubating, but sometimes not all have sedimented yet. Would a short several second spin likely to disrupt the complex binding or not, just to see the beads? 
It's better to check the beads at the time of pipetting than after incubation and centrifugation step.

 

Meanwhile, one of the reasons why the WB ended so badly may have been badly prepared run buffer (unfortunatelly, I didn't have enough experience to recognize that from the missing protein marker, so it never crossed my mind). I will rerun it to see if it actually failed that much.

[mdfenko]

the complex should survive a short spin. i ensure that the beads are evenly suspended and take equal aliquots, this way i can assume that the quantity of beads is approximately equal for all samples.

[Trof]

I settled with pipetting the whole volume up and down with a 1mL tip before adding to each sample (using the same tip). Then I leave all samples to stand in ice for a while to check visually the amount of beads (so far I didn't need to adjust it, it's more for prventing my anxiety about possible differences found a day later). I do not spin them, do not vortex them and mix after washing by slow inverting two times. The amount seems very consistent and last result looked good.

Thanks all, I think I'm slowly stopping hating them