We had the same problem trying to pull free histone proteins down from Plasma. We found that pre-clearing the plasma with protein G dynabeads helped immensely, and was very straight foward. Hope this helps.

@Michael, Please share what volume of protein G dynabeads you used against what volume of plasma for the purpose of pre-clearing ?

I am using 50 microlitre of protein G dynabeads against 100 microlitre of plasma for the purpose of IgG depletion. I also aim to immunocapture histones. But so far, pre-clearing does not seem to work properly and yet to optimise to get better results.

I will be very happy to hear from you.

Thanks !

Dear all, could you establish a nice protocol? We are also interested in pulling a protein out of mouse plasma and then analyzing by western blot. Many thanks!

Dear all, I'm very interesting by your conversation : I had the same issue. Could you send me your working protocol please?

Thank you very much!

Himanshu Chauhan : It depends on the animal the serum is from, which IgG subclass you want to clear, and the binding capacity of your beads.

Matthew B Hudson : I am working with human plasma, and interested in total IgG depletion ideally (which is impossible). I have tried number of strategies as well as ELISA kits for IgG quantification after depletion. Nothing worked well in my hands so far.

First the disclaimer: Like most things in research "it varies" so I don't want to guarantee that what works for the members of my lab will work for you.

I recently presented some work where we had data were we used a bead based capture method from human plasma (and culture media), and for some reason people have been asking me about this a lot lately. It took us a long time to do this well and is not as simple and straightforward as we initially thought it would be. And based on the questions here (and at conferences) it appears others are are at the point we were at a couple years ago. Priscilla Van den ackerveken , Jonathan D Finn , Pierre-Louis Tharaux if you are still working on this and I can be of help just let me know. For example, Pierre-Louis Tharaux we have done this in mouse plasma before as well.

Hopefully this can be helpful. I know there are numerous other threads on Researchgate asking about this or things similar, so if you posted there (or see others) just refer them here and I'll be glad to help. Hopefully that will help consolidate the discussion to one place. If it would be useful to people I could post some of files and protocols on our lab website. I've actually debated making an affordable kit for other groups to use or to just do it for other groups at an affordable rate.

Himanshu Chauhan: What are you attempting to use to capture histones (ex. IgG1 antibody, aptamer, etc.) ? Also, do you care what

Through experiments that I won't describe for the sake of time (and because we are about to try and publish them) we have determined an estimate of the total of IgG in mouse and human plasma.

Here is the average estimated amount of IgG in human plasma (hopefully this formats correctly on this website, we have an excel file).

Total IgG (mg/ml) 14.75

IgG1 (mg/ml) 7.25

IgG2 (mg/ml) 5

IgG3 (mg/ml) 0.7

IgG4 (mg/ml) 0.35

IgM (mg/ml) 1.5

IgA (mg/ml) 1.95

Also, we have tried a variety of different plasma preparations and for us plasma collected in ACD tubes and depleted of platelets works the best in our hands.

To "pre clear" you need to determine the binding capacity of the particular beads you are using, and then then determine the estimated amount of IgG in your samples (from the estimates above). Then slightly overestimate for pre-clearling. So if you want to clear total IgG from 1 ml of plasma the estimated amount of total IgG in 1 ml is 14.75. If 50 uL of your beads has a binding capacity of 10 mg IgG (I'm making this number up for the sake of simplicity, check the product info sheet on your beads for this info), then you would add 100 uL of beads. Theoretically 100 uL of those beads would bind 20 mg of total IgG.

Other tips: Dilute plasma 1:1-1:5 in PBS with 0.05% Tween 20.

Release or lyse the IgG that is (hopefully) on the beads after clearing to verify capture on the the beads. Then compare that to the 'cleared' plasma. If there is no IgG on the beads then you have a problem with your beads. If there is IgG on the beads but the plasma is still not clear of IgG then increase bead amount and/or incubation time.

I can say that it sounds like we have had a similar issue to you in the past (ie. protein G beads didn't clear the plasma IgG from plasma). Long story short, there is a lot of variability in the different bead types, brands, etc. We now custom make beads that work well. I know that's not helpful to you at this point, but I only mention that to let you know that it may not be that you are doing something incorrectly.

Matt

Matthew B Hudson thank you for the input. About an year ago, I was working with plasma extracted from fresh blood from human pregnant females. My goal was to capture cell-free nucleosomes/ cfDNA which is found in maternal blood stream just after 3-4 weeks of gestation, and quickly clears from the blood stream after 2-3 hours of delivery. I was performing ChIP on plasma (after pre-clearing) using different antibodies like H3-Pan, H2-Pan, H4, HMGN and H3K9me3 etc. I also prepared libraries and sequenced the ChIP'd DNA. The sequencing results were not good and we were not able to see desired peaks in Bioinformatic analysis. I tried many options/strategies but in the end I was not successful. Perhaps, I think it could be because of many reasons: may be the cf-nucleosomes were not stable and the immunoprecipitation didn't work as expected or pre-clearing was not enough or the amount of cf-nucleosomes/cfDNA is too less in maternal stream to be immunocaptured and further analysed.

Do you by chance have any experience with cell-free nucleosomes found freely in human blood stream ?

These days many people are working on developing kits for early cancer diagnosis using blood or for non-invasive prenatal testing.