We have a working immunoprecipitation protocol for my protein of interest, which has recoveries over 90% when using small volumes of serum (200-500 uL). We now want to scale up and immunoprecipitate more protein so it can be used in mass spectrometry. Problem is that we can't seem to get our recoveries over 25% anymore.
Fun detail is that this percentage is more or less valid despite the concentration of the protein in serum. With 10x more proteins you also get 10x more protein on your beads. So saturation of the beads is not a problem. We tried increasing the incubation time from 1 hour to overnight. This helps a bit, but still does not come close to the recoveries we're used to.
Does anyone has any tips or clues on how to continue?
If the bead saturation is not an issue, you could try to reduce the volume of your serum by centrifuge based concentration system. this might get you closer to your small scale IP conditions. You can also try several sequential IP(by changing the beads) and elute all the beads fraction at once to recover more protein.
Hi Alexander,
I have done that few years ago and I would recommend that you spin the serum first to get rid of macromolecules and fat then you run your samples into Amicon tubes (containing membrane) where you can choose which molecules to pass through the membrane (perhaps those that are less than 3000 Da). These tubes are available in various sizes. I have used 1.0 ml tubes to concentrate my sample. The centrifugations time could take 20-60 min. These tubes are available from Millipore.
Good Luck
If you know the approximate size of the proteins, then cut off filters is one way to reduce the volume. secondly you can precipitate the serum proteins by ammonium sulfate, and then dialyze it for some time in IP buffer (you have to check this in the small scale first that you are getting your proteins precipitated and not loosing them). this will very quickly give you the best concentrated proteins, and if you are careful for selective precipitation, you can get rid off the serum albumin, which is huge and gives u background, also the IgG in the serum. Thirdly after preclearing for the macromolecules, then dialyze the serum with sequentially high percentage of glycerol, until u get the desired volume, later you can dilute finally with IP buffer.
Multiple IP is OK but since the effective concentration is getting lower everytime, so the kinetics of binding also gets lower.
Hope this helps.
Try ammonium sulfate first, in 1 ml serum.
I would suggest that first concentrate your protein by using the molecular cut-off filters of your interest (which are available in various sizes, Millipore) and centrifuge or precipitate using ammonium sulphate. Later carry on with immunoprecipation assay using beads to recover more protein. Hope it helps.
Thank you all for your answers!
We will look into the Amicon tubes as many have suggested. Firstly, however, we will try to use centrifuge concentration to increase the concentration of the protein. We will also see if increasing the amount of beads, or repeatedly IP'ing of the same sample will work.
Alexander,
Would you share that protocol for small-scale IP that works?
thank you,
Peter
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